Injectable Cryogel Vaccine Devices and Methods of Use Thereof

ABSTRACT

The invention provides polymer compositions for cell and drug delivery.

RELATED APPLICATIONS

This application is continuation of U.S. application Ser. No. 14/166,689filed Jan. 28, 2014, which is a continuation-in-part of U.S. applicationSer. No. 14/112,096, which is a national stage application, filed under35 U.S.C. § 371, of International Application No. PCT/US2012/035505filed Apr. 27, 2012, which claims the benefit of priority under 35U.S.C. § 119(e) to U.S. Provisional Application No. 61/480,237 filedApr. 28, 2011. This application also claims the benefit of priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/915,985filed Dec. 13, 2013 and U.S. Provisional Application No. 61/757,509filed Jan. 28, 2013. The contents of each of these applications areincorporated herein by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. Government support under Grant NumbersR01 DE013349, 5R01 DE019917-03, and R01 EB015498 from the NationalInstitutes of Health and Award Number ECS-0335765 from the NationalScience Foundation. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named“29297-084C02US_Sequence_Listing.txt”, which was created on Jun. 8, 2017and is 2,530 bytes in size, is hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to polymer-containing devices for drug and celldelivery systems.

BACKGROUND

Cancer is a devastating disease with a high mortality rate that canaffect nearly any organ in the body. Although treatment options existfor certain cancers, these options are limited in terms of efficacy,safety, and applicability to a wide range of cancer types. Thus, thereis a need for more effective, safe, and widely-applicable cancertreatments. There is also a need for methods of preventing cancer.

Three-dimensional polymer-containing devices, such as scaffold matrices,have been used for a number of applications, including tissueregeneration/repair and cell transplantation. For example, porous andbiodegradable polymer scaffolds have been utilized as a structuralsupporting matrix or as a cell adhesive substrate for cell-based tissueengineering. However, a major side effect of the surgical implantationof three dimensional scaffolds is the trauma created by physicians whiletreating patient illness. In particular, current technologies for thesurgical implantation of three dimensional scaffolds involve incisionsthat lead to patient pain, bleeding, and bruising. As such, there is apressing need in the art to develop less invasive structuredpolymer-containing devices.

This invention addresses these needs.

SUMMARY OF THE INVENTION

The invention features injectable hydrogel/cryogel-based vaccinesgreatly increase the efficacy of vaccine therapy for many cancer types,including melanoma and breast cancer. These vaccines provide much neededhope for patients with these fatal diseases.

The invention provides a cryogel sponge-like vaccine that has severaladvantages as a prophylactic cancer vaccine (e.g., for melanoma orbreast cancer). First, the vaccine does not require expensive,time-consuming preparation and expansion of cells in some versions.Second, it is safely and easily administered with subcutaneousinjection. Third, the diversity of native cancer antigens available fromthe use of whole cancer cells, conservation of cancer-specific cellsurface antigens, and the ability to recruit, house, and activate immunecells, demonstrates the potential for using cryogel-based vaccines todevelop safer and more effective cellular cancer vaccines with long-termprotective benefits.

The present invention also provides compositions and aminimally-invasive method of injecting preformed large macroporouspolymer-based hydrogels (e.g., cryogels) that are loaded with cargo suchas cells and/or therapeutics such as small molecule compounds,proteins/peptides (e.g., antigens to which an immune response isdesired), or nucleic acids. Hydrogel (also called aquagel) is a networkof polymer chains that are hydrophilic, and are sometimes found as acolloidal gel in which water is the dispersion medium. Hydrogels arehighly absorbent (they can contain over 99% water) natural or syntheticpolymers that possess a degree of flexibility very similar to naturaltissue, due to their significant water content. Unlike conventionalhydrogels, a unique characteristic of the devices described herein isthat when an appropriate shear stress is applied, the deformablehydrogel is dramatically and reversibly compressed (up to 95% of itsvolume), resulting in injectable macroporous preformed scaffolds. Thisproperty allows the devices to be delivered via syringe with highprecision to target sites.

Accordingly, the invention features a cell-compatible and optionally,cell-adhesive, highly crosslinked hydrogel (e.g., cryogel) polymercomposition comprising open interconnected pores, wherein the hydrogel(e.g., cryogel) is characterized by shape memory following deformationby compression or dehydration. The device has a high density of openinterconnected pores. Also, the hydrogel (e.g., cryogel) comprises acrosslinked gelatin polymer or a crosslinked alginate polymer.

Examples of polymer compositions from which the cryogel is fabricatedinclude alginate, hyaluronic acid, gelatin, heparin, dextran, carob gum,PEG, PEG derivatives including PEG-co-PGA and PEG-peptide conjugates.The techniques can be applied to any biocompatible polymers, e.g.collagen, chitosan, carboxymethylcellulose, pullulan, polyvinyl alcohol(PVA), Poly(2-hydroxyethyl methacrylate) (PHEMA),Poly(N-isopropylacrylamide) (PNIPAAm), or Poly(acrylic acid) (PAAc). Forexample, the composition comprises an alginate-based hydrogel/cryogel.In another example, the composition comprises a gelatin-basedhydrogel/cryogel.

In some embodiments, the invention also features gelatin scaffolds,e.g., gelatin hydrogels such as gelatin cryogels, which are acell-responsive platform for biomaterial-based therapy. Gelatin is amixture of polypeptides that is derived from collagen by partialhydrolysis. These gelatin scaffolds have distinct advantages over othertypes of scaffolds and hydrogels/cryogels. For example, the gelatinscaffolds of the invention support attachment, proliferation, andsurvival of cells and are degraded by cells, e.g., by the action ofenzymes such as matrix metalloproteinases (MMPs) (e.g., recombinantmatrix metalloproteinase-2 and -9).

Prefabricated gelatin cryogels rapidly reassume their original shape(“shape memory”) when injected subcutaneously into a subject (e.g., amammal such as a human, dog, cat, pig, or horse) and elicit little or noharmful host immune response (e.g., immune rejection) followinginjection. In some examples, gelatin hydrogels are loaded withgranulocyte-macrophage colony-stimulating factor (GM-CSF). Controlledrelease of GM-CSF from gelatin cryogels results in significantinfiltration of the scaffold by immune cells and promotes matrixmetalloproteinase production, leading to cell-mediated degradation ofthe cryogel matrix.

In some embodiments, the hydrogel (e.g., cryogel) comprises polymersthat are modified, e.g., sites on the polymer molecule are modified witha methacrylic acid group (methacrylate (MA)) or an acrylic acid group(acrylate). Exemplary modified hydrogels/cryogels are MA-alginate(methacrylated alginate) or MA-gelatin. In the case of MA-alginate orMA-gelatin, 50% corresponds to the degree of methacrylation of alginateor gelatin. This means that every other repeat unit contains amethacrylated group. The degree of methacrylation can be varied from 1%to 90%. Above 90%, the chemical modification may reduce solubility ofthe polymer water-solubility.

Polymers can also be modified with acrylated groups instead ofmethacrylated groups.

The product would then be referred to as an acrylated-polymer. Thedegree of methacrylation (or acrylation) can be varied for mostpolymers. However, some polymers (e.g. PEG) maintain theirwater-solubility properties even at 100% chemical modification. Aftercrosslinking, polymers normally reach near complete methacrylate groupconversion indicating approximately 100% of cross-linking efficiency.For example, the polymers in the hydrogel are 50-100% crosslinked(covalent bonds). The extent of crosslinking correlates with thedurability of the hydrogel. Thus, a high level of crosslinking (90-100%)of the modified polymers is desirable.

For example, the highly crosslinked hydrogel/cryogel polymer compositionis characterized by at least 50% polymer crosslinking (e.g., 75%, 80%,85%, 90%, 95%, 98%). The high level of crosslinking confers mechanicalrobustness to the structure. However, the % crosslinking is generallyless than 100%. The composition is formed using a free radicalpolymerization process and a cryogelation process. For example, thecryogel is formed by cryopolymerization of methacrylated gelatin ormethacrylated alginate. In some cases, the cryogel comprises amethacrylated gelatin macromonomer or a methacrylated alginatemacromonomer concentration of 1.5% (w/v) or less (e.g., 1.5%, 1.4%,1.3%, 1.2%, 1.1%, 1% 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% orless). For example, the methacrylated gelatin or alginate macromonomerconcentration is about 1% (w/v).

In some embodiments, crosslinked gelatin hydrogels/cryogels are formedby modification of gelatin with pendant methacrylate groups. Forexample, crosslinking occurs via radical polymerization. In someexamples, 2-6% (e.g., 3-4%) of the amino acid composition of gelatin islysine. In some cases, lysine in the gelatin is converted to reactivemethacrylate groups. In some cases, 70-90% (e.g., 80%) of the lysine inthe gelatin is converted to reactive methacrylate groups. These reactivemethacrylate groups on the gelatin are then crosslinked, e.g., byradical polymerization. In some embodiments, the gelatin polymers of theinvention (e.g., crosslinked by radical polymerization) contain agreater number of crosslinks compared to a gelatin polymer incubated atroom temperature without radical polymerization (e.g., withoutmodification by methacrylate).

The cryogel comprises at least 75% pores, e.g., 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% or more pores. The pores areinterconnected. Interconnectivity is important to the function of thecomposition, as without interconnectivity, water would become trappedwithin the gel. Interconnectivity of the pores permits passage of water(and other compositions such as cells and compounds) in and out of thestructure. In a fully hydrated state, the composition comprises at least90% water (e.g., between 90-99%, at least 92%, 95%, 97%, 99%, or more)water. For example, at least 90% (e.g., at least 92%, 95%, 97%, 99%, ormore) of the volume of the cryogel is made of liquid (e.g., water)contained in the pores. In a compressed or dehydrated hydrogel, up to50%, 60%, 70% of that water is absent, e.g., the cryogel comprises lessthan 25% (20%, 15%, 10%, 5%, or less) water.

The cryogels of the invention comprises pores large enough for a cell totravel through. For example, the cryogel contains pores of 20-500 am indiameter, e.g., 20-300 am, 30-150 am, 50-500 am, 50-450 am, 100-400 am,200-500 am. In some cases, the hydrated pore size is 1-500 am (e.g.,10-400 am, 20-300 am, 50-250 lam).

Injectable cryogels can also be produced in a form in whichpharmaceuticals or other bioactive substances (e.g. growth factors, DNA,enzymes, peptides, drugs, etc) are incorporated for controlled drugdelivery.

In some embodiments, injectable cryogels are further functionalized byaddition of a functional group chosen from the group consisting of:amino, vinyl, aldehyde, thiol, silane, carboxyl, azide, alkyne.Alternatively, the cryogel is further functionalized by the addition ofa further cross-linker agent (e.g. multiple arms polymers, salts,aldehydes, etc). The solvent can be aqueous, and in particular acidic oralkaline. The aqueous solvent can comprise a water-miscible solvent(e.g. methanol, ethanol, DMF, DMSO, acetone, dioxane, etc).

The cryo-crosslinking takes place in a mold and the injectable cryogelscan be degradable. The pore size can be controlled by the selection ofthe main solvent used, the incorporation of a porogen, the freezingtemperature and rate applied, the cross-linking conditions (e.g. polymerconcentration), and also the type and molecule weight of the polymerused.

In some examples, the composition comprises a cell adhesion compositionchemically linked, e.g., covalently attached, to the polymer. Forexample, the cell adhesion composition comprises a peptide comprising anRGD amino acid sequence. In other examples, the cryogel composition(e.g., gelatin) has cell-adhesive properties. In some cases, the cryogel(e.g., gelatin cryogel) is not modified with a cell adhesive molecule,such as arginine-glycine-aspartate (RGD).

For cell therapy, the composition comprises a eukaryotic cell in one ormore of the open interconnected pores. For example, the eukaryotic cellcomprises a live attenuated cancer cell (e.g., irradiated cell acts ascancer antigen). Exemplary cancer cells include but are not limited tomelanoma cells, breast cancer cells, central nervous system (CNS) cancercells, lung cancer cells, leukemia cells, multiple myeloma cells, renalcancer cells, malignant glioma cells, medulloblastoma cells, coloncancer cells, stomach cancer cells, sarcoma cells, cervical cancercells, ovarian cancer cells, lymphoma cells (e.g., Non-Hodgkin'slymphoma cells), pancreatic cancer cells, prostate cancer cells, thyroidcancer cells, rectal cancer cells, endometrial cancer cells, and bladdercancer cells. In another example, the cell is a stem cell, progenitor,or other cell that contributes to tissue repair or regeneration.

The hydrogel/cryogel, if to be used to transplant cells, comprises poresto permit the structure to be seeded with cells and to allow the cellsto proliferate and migrate out to the structure to relocate to bodilytissues such as the injured or diseased muscle in need of repair orregeneration. For example, cells are seeded at a concentration of about1×10³ to 1×10⁸ cells/mL (e.g., about 5×10³ to 5×10⁷ cells/mL, or about1×10⁴ to 1×10⁷ cells/mL) and are administered dropwise onto a driedhydrogel/cryogel device. For example, the cells are seeded in a cryogelhaving a volume (when hydrated) of 1-500 uL (e.g., 10-250 uL, 20-100 uL,or 40-60 uL, or about 50 uL). The dose of the gel/device to be deliveredto the subject is scaled depending on the magnitude of the injury ordiseased area, e.g., one milliliter of gel for a relatively small defectand up to 50 mL of gel for a large wound. Preferably, thehydrogel/cryogel comprises macropores, e.g., pores that arecharacterized by a diameter of 2 μm-1 mm. The average pore sizecomprises 200 μm. Cells can move into and out of the cryogel via theopen interconnected pores as a typical cell comprises a diameter orabout 20 μm. The gel delivery devices are suitable for treatment ofhuman beings, as well as animals such as horses, cats, or dogs.

Optionally, the device comprises a biomolecule in one or more of theopen interconnected pores. Biomolecules include small molecule compounds(e.g., less than 1000 daltons in molecular mass), nucleic acids,proteins or fragments thereof, peptides. Biomolecules are purifiednaturally-occurring, synthetically produced, or recombinant compounds,e.g., polypeptides, nucleic acids, small molecules, or other agents. Forexample, the compositions include a chemotactic protein, granulocytemacrophage-colony stimulating factor (GM-CSF), pathogen-associatedmolecular patterns (PAMPs) such as CpG oligodeoxynucleotide (CpG-ODN),and cancer antigens or other antigens. The compositions, e.g., antigens,described herein are purified. Purified compounds/molecules are at least60% by weight (dry weight) the compound/molecule of interest.Preferably, the preparation is at least 75%, more preferably at least90%, and most preferably at least 99%, by weight the compound ofinterest. Purity is measured by any appropriate standard method, forexample, by column chromatography, polyacrylamide gel electrophoresis,or HPLC analysis. Exemplary biomolecules include GM-CSF, large nucleicacid compositions such as plasmid DNA, and smaller nucleic acidcompositions such as CpG-ODN. Other exemplary biomolecules includecancer antigens, e.g., derived from one or more of the cancers listedabove, e.g., purified cancer antigens.

For example, the cryogel contains 1-10 ug of GM-CSF. In some examples,the cryogel has a volume of 1-500 uL (e.g., 10-250 uL, 20-100 uL, or40-60 uL, or about 50 uL). In some cases, the nucleic acid biomoleculecomprises a CpG nucleic acid oligonucleotide. For example, the CpG ODNis incorporated into the cryogel by mixing free CpG ODN with crosslinkedgelatin. In other cases, the CpG ODN is incorporated into the cryogel byi) condensing the CpG ODN into nanoparticles to form CpG ODNcondensates, and ii) crosslinking the gelatin with the CPG ODNcondensates. Alternatively, the CpG ODN is covalently bound to thecryogel or the CpG ODN is ionically bonded to the cryogel.

In some examples, the device recruits a cell into the cryogel upon andafter injection into a subject. The cell is recruited into one or moreof the open interconnected pores of the cryogel. For example, therecruited cell comprises an immune cell, e.g., an antigen presentingcell (APC), a granulocyte, a macrophage, T cell (e.g., cytotoxic T cellor regulatory T cell), B cell, natural killer (NK) cell, or dendriticcell (DC). For example, the T cell is a cytotoxic T cell or a regulatoryT cell. In some cases, the cryogel is degraded by one or more recruitedcells (e.g., by a protease, such as a matrix metalloproteinase,expressed by the recruited cell). The cryogel is degraded at a ratedependent on the number of cells recruited into the cryogel.

Preferably, the cryogel compositions are injectable through a hollowneedle. For example, the scaffold composition is injectable through a16-gauge, an 18-gauge, a 20-gauge, a 22-gauge, a 24-gauge, a 26-gauge, a28-gauge, a 30-gauge, a 32-gauge, or a 34-gauge needle. Upon compressionor dehydration, the composition maintains structural integrity and shapememory properties, i.e., after compression or dehydration, thecomposition regains its shape after it is rehydrated or the shear forcesof compression are removed/relieved. The scaffold composition alsomaintains structural integrity in that it is flexible (i.e., notbrittle) and does not break under sheer pressure.

In some examples, the cryogel/device is between 0.01 mm³ and 100 mm³.For example, the cryogel/device is between 1 mm³ and 75 mm³, between 5mm³ and 50 mm³, between 10 mm³ and 25 mm³. Preferably, thecryogel/device is between 1 mm³ and 10 mm³ in size.

The shape of the cryogel is dictated by a mold and can thus take on anyshape desired by the fabricator, e.g., various sizes and shapes (disc,cylinders, squares, strings, etc.) are prepared by cryogenicpolymerization. Injectable cryogels can be prepared in themicrometer-scale to millimeter-scale. Volume varies from a few hundredμm³ (e.g., 100 μm³) to over 100 mm³. An exemplary scaffold compositionis between 100 μm³ to 100 mm³ in size (e.g., between 1 mm³ and 10 mm³ insize). In another example, the cryogel is defined by volume. Forexample, the cryogel scaffold composition comprises 5-100 uL (e.g., 25μL) in volume in a hydrated state. The gels are hydrated in an aqueousmedium. Exemplary cryogel compositions are typically in the range of10-70 μL in volume and may be larger or smaller depending on the use andsite to be treated.

The cryogel acts as a sponge. The cryogels are sterilized. In someapplications, the cryogels are hydrated, loaded with cells or othercompounds (e.g., small molecules and other compounds, nucleic acids, orproteins/peptides) and loaded into a syringe or other deliveryapparatus. For example, the syringes are prefilled and refrigerateduntil use. In another example, the cryogel is dehydrated, e.g.,lyophylized, optionally with a drug or other compound loaded in the geland stored dry or refrigerated. Prior to administration, thecryogel-loaded syringe or apparatus is contacted with a solutioncontaining cells and/or other compounds to be delivered. For example,the barrel of the cryogel pre-loaded syringe is filled with aphysiologically-compatible solution, e.g., phosphate-buffered saline(PBS). In practice, the cryogel is administered to a desired anatomicalsite followed by the volume of solution, optionally containing otheringredients, e.g., cells or therapeutic compounds. For example, a 25 μLcryogel is administered with approximately 200 μL of solution. Thecryogel is then rehydrated and regains its shape integrity in situ. Thevolume of PBS or other physiologic solution administered followingcryogel placement is generally about 10 times the volume of the cryogelitself.

Cell viability is minimally affected or unaffected by the shear thinningprocess, and gel/cell constructs stay fixed at the point ofintroduction. As such, these gels are useful for the delivery of cellsand other compounds to target biological sites in therapeutic methodssuch as tissue regeneration (cell therapy, drug delivery) efforts.

The cryogel also has the advantage that, upon compression, the cryogelcomposition maintains structural integrity and shape memory properties.For example, the cryogel is injectable through a hollow needle. Forexample, the cryogel returns to its original geometry after travelingthrough a needle (e.g., a 16 gauge (G) needle, e.g., having an 1.65 mminner diameter). Other exemplary needle sizes are 16-gauge, an 18-gauge,a 20-gauge, a 22-gauge, a 24-gauge, a 26-gauge, a 28-gauge, a 30-gauge,a 32-gauge, or a 34-gauge needle.

The polymer chains of the hydrogel/cryogel are covalently crosslinkedand/or oxidized. Such hydrogels are suitable for minimally-invasivedelivery. Prior to delivery into the human body, such a hydrogel islyophylized and compressed prior to administration to a subject for theregeneration of muscle tissue. Minimally-invasive delivery ischaracterized by making only a small incision into the body. Forexample, the hydrogel is administered to a muscle of a subject using aneedle or angiocatheter.

Injectable cryogels have been designed to pass through a hollowstructure, e.g., very fine needles, such as 18-30 G needles, as a tissuefiller for applications in cosmetic surgery, for tissue augmentation,and tissue repair which may be due to injury caused by disease andexternal trauma. The injectable cryogels may be molded to a desiredshape, in the form of rods, square, disc, spheres, cubes, fibers, foams.In some situations, the injectable cryogels can be used as scaffolds forcell incorporation. In some cases, the cryogel comprises the shape of adisc, cylinder, square, rectangle, or string. For example, the cryogelcomposition is between 100 μm³ to 100 mm³ in size, e.g., between 1 mm³to 50 mm³ in size. For example, the cryogel composition is between 1 mmin diameter to 50 mm in diameter (e.g., around 5 mm). Optionally, thethickness of the cryogel is between 0.2 mm to 50 mm (e.g., around 2 mm).The formed cryogel is mixed with cells to provide tissue engineeredproducts, or can be used as a bio-matrix to aid tissue repair or tissueaugmentation. The incorporated cells can be any mammalian cells (e.g.stem cells, fibroblasts, osteoblasts, chrondrocytes, immune cells, etc).

The cryogel also has the advantage of inducing a minimal adverse hostresponse (e.g., minimal immune rejection and/or minimal harmful acuteinflammation) after injection into a subject.

Therapeutic and cosmetic uses are described throughout thespecification. Exemplary applications include use as a dermal filler, indrug delivery, as a wound dressing, for post surgical adhesionprevention, and for repair and/or regenerative medical applications suchas cell therapy, gene therapy, tissue engineering, immunotherapy.

For example, a method for repairing, regenerating, or restructuring atissue comprises administering to a subject the device/cryogelcomposition described above. If the cryogel contains cells, the cellsretain their viability after passage through the syringe or deliveryapparatus, cells proliferate in the device/cryogel, then leave thecryogel composition to function outside of the gel and in the bodilytissues of the recipient subject. For example, the cryogel isadministered subcutaneously as a dermal filler, thereby restructuringthe tissue, e.g., dermal tissue. In another example, the cryogel devicecomprises a stem cell and the composition/device is administered to adamaged or diseased tissue of a subject, thereby repairing orregenerating the tissue, e.g., muscle, bone, kidney, liver, heart,bladder, ocular tissue or other anatomic structures.

In another example, the cryogel compositions are used in a method fordelivering genetic material to a tissue, e.g., to deliver plasmid DNA.

The invention also features a method for eliciting an immune response,comprising administering to a subject a cryogel composition describedherein, where the composition comprises a microbial pathogen, microbialantigen, cancer cell lysate, cancer antigen, or cancer cell to which animmune response is elicited. For example, cancer cells include but arenot limited to cells from a melanoma, a breast cancer, a lung cancer, alymphoma (e.g., Non-Hodgkin's lymphoma cells), a leukemia, a stomachcancer, a liver cancer, a central nervous system cancer, a sarcoma, acentral nervous system (CNS) cancer, a multiple myeloma, a renal cancer,a malignant glioma, a medulloblastoma, a colon cancer, a cervicalcancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, athyroid cancer, a rectal cancer, an endometrial cancer, a uterinecancer, and a bladder cancer. For example, a cancer lysate or cancerantigen is derived from a cancer cell described herein.

For example, a microbial pathogen includes but is not limited to afungus, a bacterium (e.g., Staphylococcus species, Staphylococcusaureus, Streptococcus species, Streptococcus pyogenes, Pseudomonasaeruginosa, Burkholderia cenocepacia, Mycobacterium species,Mycobacterium tuberculosis, Mycobacterium avium, Salmonella species,Salmonella typhi, Salmonella typhimurium, Neisseria species, Brucellaspecies, Bordetella species, Borrelia species, Campylobacter species,Chlamydia species, Chlamydophila sepcies, Clostrium species, Clostriumbotulinum, Clostridium difficile, Clostridium tetani, Helicobacterspecies, Helicobacter pylori, Mycoplasma pneumonia, Corynebacteriumspecies, Neisseria gonorrhoeae, Neisseria meningitidis Enterococcusspecies, Escherichia species, Escherichia coli, Listeria species,Francisella species, Vibrio species, Vibrio cholera, Legionella species,or Yersinia pestis), a virus (e.g., adenovirus, Epstein-Barr virus,Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Herpes simplexvirus type 1, 2, or 8, human immunodeficiency virus, influenza virus,measles, Mumps, human papillomavirus, poliovirus, rabies, respiratorysyncytial virus, rubella virus, or varicella-zoster virus), a parasiteor a protozoa (e.g., Entamoeba histolytica, Plasmodium, Giardia lamblia,Trypanosoma brucei, or a parasitic protozoa such as malaria-causingPlasmodium). For example, a microbial antigen is derived from amicrobial cell described herein.

For example, the cancer cell or microbial pathogen cell is a liveattenuated (e.g., /irradiated) cell. In the case of microbial pathogens,heat-killed bacteria are optionally used. In some cases, the cell isobtained from the patient to be treated, e.g., by biopsy, attenuated,and then used as a component of the device.

In some examples, the cancer cell is a melanoma cell (e.g., B16-F10cancer cell or patient-derived, autologous attenuated cell) or a breastcancer cell (e.g., a HER-2/neu-overexpressing breast cancer cell such asa patient-derived, autologous cancer cell). In some examples, thecryogel composition contains a protein or polypeptide (e.g., recombinantprotein) or a plasmid DNA encoding for the protein or polypeptidederived from a microbial pathogen or cancer cell described herein. Thecryogel composition (e.g., in the form of a device) is administeredprophylactically or therapeutically.

In some embodiments, the device is injected into the subject once everyday to once every 10 years (e.g., once every day, once every week, onceevery two weeks, once every month, once every two months, once every 3months, once every 4 months, once every 5 months, once every 6 months,once every year, once every 2 years, once every 3 years, once every 4years, once every 5 years, once every 6 years, once every 7 years, onceevery 8 years, or once every 10 years). In other examples, the device isinjected into the subject once to 5 times (e.g., one time, twice, 3times, 4 times, 5 times, or more as clinically necessary) in thesubject's lifetime.

The injected device comprises at least 0.5×10⁶ (e.g., at least 0.75×10⁶,at least 1×10⁶, at least 1.5×10⁶, at least 2×10⁶, at least 5×10⁶, atleast 10×10⁶, or more) immune cells at least 1 day (e.g., at least 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,19 days, 20 days, or more) after injection into the subject.

In some cases, the injected device induces an increase in the number ofimmune cells in a lymph node (e.g., draining lymph node) or spleen atleast 1 day (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more days) after injection into the subject.

Exemplary immune cells include DC (e.g., CD11b+, CD11c+, plasmacytoid,and CD8+DC), T cells (e.g., cytotoxic T cells and/or Treg cells), Bcells, macrophages, granulocytes, and natural killer cells. For example,the immune cells comprise CD3+ T cells, CD8+ T cells, and/or FoxP3+ Tregcells. In some cases, the ratio of CD8+ effector T cells to FoxP3+ Tregcells is at least 0.5-fold (at least 0.75-fold, at least 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 7-fold, 10-fold, 15-fold, or more). In someexamples, at least 40% (e.g., at least 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, or more) of the immune cells are plasmacytoid DC. Inother examples, at least 20% (e.g., at least 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, or more) of the immune cells are CD8+DC, orCD11c+CD141+ human DC. See, e.g., Bachem et al. J. Exp. Med.207(2010):1273-81; and Lauterbach et al. J. Exp. Med. 207(2010):2703-17,incorporated herein by reference.

The injected device comprises 10⁷ or fewer (e.g., 1×10⁷, 8×10⁶, 5×10⁶,4×10⁶, 3×10⁶, 2×10⁶, 1×10⁶, 5×10⁵, 2×10⁵, 1×10⁵, 1×10⁴, or fewer) cells(e.g., immune cells) at least 15 days (e.g., at least 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days) afterinjection. Exemplary immune cells are described above.

The injected device and/or tissue surrounding the device (e.g., within10 cm of the injected device, such as within 10 cm, 9 cm, 8 cm, 7 cm, 6cm, 5 cm, 4 cm, 3 cm, 2 cm, 1 cm, or fewer) comprises an elevated levelof a cytokine compared to the level of the cytokine at a site in thesubject more than 10 cm away from the injected device (e.g., at adifferent site in the body), e.g., more than 10 cm away from a boundary,edge, or center of the injected device. Alternatively, the injecteddevice and/or tissue surrounding the device (e.g., within 10 cm of theinjected device, such as within 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4cm, 3 cm, 2 cm, 1 cm, or fewer) comprises an elevated level of acytokine compared to the level of the cytokine in the subject at thesite of injection prior to injection. In some examples, the injecteddevice and/or tissue surrounding the device (e.g., within 10 cm of theinjected device, such as within 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4cm, 3 cm, 2 cm, 1 cm, or fewer) comprises an elevated level of acytokine compared to the level of the cytokine in an untreated subject.The level of the cytokine is elevated by at least 1.5-fold (e.g., atleast 1.8-fold, 2-fold, 4-fold, 5-fold 7-fold, 10-fold, 15-fold,20-fold, or more).

The cytokine comprises RANTES (regulated on activation, normal T cellexpressed and secreted) (also called Chemokine (C—C motif) ligand 5(CCL5)), eotaxin, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9,IL-10, IL-12, IL-13, IL-17, GM-CSF, macrophage inflammatory protein-1α(MIP-1α), macrophage inflammatory protein-1β ((MIP-1β),keratinocyte-derived chemokine (KC), tumor necrosis factor-α (TNF-α),granulocyte-colony stimulating factor (G-CSF), interferon-α (IFN-α),interferon-γ (IFN-γ), or monocyte chemotactic protein-1 (MCP-1).

In some embodiments, the subject to be administered a cryogel/device ofthe invention does not have a cancer, has not been diagnosed with acancer, or has been diagnosed with a cancer.

In some cases, the device increases the survival time of a subjectdiagnosed with a cancer by at least 1 month (e.g., at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or 11 months or more, or at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 years or more) compared to thesurvival time of an untreated subject. Increased survival time isdetermined by comparing the prognosis for survival in the subject from atime period prior to administration of the device to the prognosis forsurvival in the subject following administration of the device, whereinan increase in predicted survival time indicates that the treatmentincreased survival of the subject following administration of thedevice.

In other cases, the device stabilizes the size (e.g., volume, mass, orlongest diameter) of an existing tumor, thereby preventing diseaseprogression. For example, the size of an existing tumor afteradministration of the device remains within 30% (e.g., within 25% 20%,15%, 10%, 5%, 2.5%, 1%, or less) of the original size beforeadministration of the device. Alternatively, the device decreases thesize (e.g., volume, mass, or longest diameter) of an existing tumor. Forexample, the device decreases the size (e.g., volume, mass, or longestdiameter) of an existing tumor by at least 1.5-fold (e.g., at least2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,15-fold, 20-fold, or more) compared to the size of the tumor prior toinjection of the device.

The vaccine device of the invention remains effective in eliciting theimmune response at least 100 days (e.g., at least 120 days, 150 days,180 days, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years,8 years 9 years, 10 years or more) after injection.

The device is injected, e.g., into the subcutis of a subject. In otherexamples, the device is injected intradermally, intramuscularly, into anorgan, or directly into a tumor. The device is injected into at least 1site (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sites) in thesubject.

In some examples, the device reduces the rate of tumor growth in thesubject compared to the rate of tumor growth in an untreated subject.For example, the device reduces the rate of tumor growth in the subjectby at least 2-fold compared to the rate of tumor growth in an untreatedsubject. For example, the device reduces the rate of tumor growth in thesubject by at least 2-fold (e.g., at least 2-fold, 3-fold, 4-fold,5-fold, 7-fold, 10-fold, 15-fold, or more) compared to the rate of tumorgrowth in a subject administered with a hydrogel lacking cancer cells(e.g., attenuated cancer cells).

Bioactive factors such as polynucleotides, polypeptides, or other agents(e.g., antigens) are purified and/or isolated. Specifically, as usedherein, an “isolated” or “purified” nucleic acid molecule,polynucleotide, polypeptide, or protein, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or chemical precursors or other chemicals when chemicallysynthesized. Purified compounds are at least 60% by weight (dry weight)the compound of interest. Preferably, the preparation is at least 75%,more preferably at least 90%, and most preferably at least 99%, byweight the compound of interest. For example, a purified compound is onethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w)of the desired compound by weight. Purity is measured by any appropriatestandard method, for example, by column chromatography, thin layerchromatography, or high-performance liquid chromatography (HPLC)analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA)or deoxyribonucleic acid (DNA)) is free of the genes or sequences thatflank it in its naturally-occurring state. Purified also defines adegree of sterility that is safe for administration to a human subject,e.g., lacking infectious or toxic agents.

Similarly, by “substantially pure” is meant that a nucleotide,polypeptide, or other compound has been separated from the componentsthat naturally accompany it. Typically, the nucleotides and polypeptidesare substantially pure when they are at least 60%, 70%, 80%, 90%, 95%,99%, or even 100%, by weight, free from the proteins andnaturally-occurring organic molecules with they are naturallyassociated. Examples include synthesized compounds, recombinantcompounds (e.g., peptides, proteins, nucleic acids) or purifiedcompounds, e.g., purified by standard procedures includingchromatographic methods.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybridgene, i.e., a gene encoding a fusion protein. Isolated nucleicacid molecules according to the present invention further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publishedforeign patents and patent applications cited herein are incorporatedherein by reference. Genbank and NCBI submissions indicated by accessionnumber cited herein are incorporated herein by reference. All otherpublished references, documents, manuscripts and scientific literaturecited herein are incorporated herein by reference. In the case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of four photomicrographs showing injectablealginate-based hydrogel systems. Rhodamine-labeled 1% methacrylated(MA)-alginate gels with various sizes and shapes (disc, cylinders,squares, etc.) were prepared by cryogenic polymerization. Square shapeinjectable scaffolds are shown. Fluorescent macroscopic gels suspendedin 0.2 mL of phosphate buffered saline (PBS) were injected via 16-gaugediameter needles with a complete geometric restoration as illustrated inthe microscopy image before and after injection.

FIG. 2 is a line graph demonstrating stress vs. strain curves forconventional nanoporous and macroporous 1% rhodamine-labeled MA-alginategels subjected to compression tests. In contrast to the brittle natureof the conventional nanoporous gels, alginate cryogels have the abilityto withstand reversibly large deformation while keeping their structuralintegrity and shape memory properties.

FIG. 3A is a fluorescence photograph showing minimally invasivesubcutaneous injection of macroporous scaffolds into the lower back ofmice. FIG. 3B is a photograph showing hydrogel localization aftersubcutaneous injection of preformed rhodamine-labeled 1% MA-alginategels (4 mm×4 mm×1 mm) in the subcutis of a mouse after 3 days. FIG. 3Cis a photograph showing merged phase-contrast and fluorescence of asubcutaneously injected rhodamine-labeled alginate macroporous scaffoldwith restoration of geometry after placement. FIG. 3D is a photograph ofa subcutaneously injected rhodamine-labeled alginate macroporousscaffold with restoration of geometry. Dashed lines denote square-shapedgeometry restoration of inserted shape-defined scaffolds. FIG. 3E is aline graph showing in vivo sustained release profiles of crosslinked(chemically anchored) or encapsulated (physically entrapped)rhodamine-labeled bovine serum albumin (BSA) to injected cryogels. Upondissection 3 days post-injection, rhodamine-labeled gels recovered theirsquare shape features, had soft consistencies, and were integrated intothe surrounding tissues. Values represent mean and standard deviation(n=4).

FIGS. 4A-4F are a series of photographs showing that injectablepre-seeded scaffolds promote in situ localization of bioluminescent B16cells. FIG. 4A is a photograph showing alginate cryogel scaffolds(white) and rhodamine-labeled alginate scaffolds (pink). BioluminescenceB16-F10 cells were seeded on 1% RGD-modified MA-Alginate cryogels at aconcentration of 200×10³ cells/scaffold. Luciferase transected melanomacells were cultured for 6 hr into rhodamine-labeled alginate cryogelsbefore injection into mice. FIG. 4B is a photograph showing optical liveimaging to demonstrate that macroporous alginate gels are suitable forhomogenous encapsulation and distribution of bioluminescent B16 cells.FIG. 4C is a photograph showing scanning electron microscope (SEM)imaging to demonstrate that macroporous alginate gels are suitable forhomogenous encapsulation and distribution of bioluminescent B16 cells.FIG. 4D is a photograph showing live fluorescence imaging ofsubcutaneous injections of gels. FIG. 4E is a photograph showing livefluorescence imaging of subcutaneous injections of gels at 2 dayspost-injection. FIG. 4F is a photograph showing live fluorescenceimaging of subcutaneous injections of gels at 9 days post-injection.Bioluminescent B16-cells were visualized by live imaging. Arg-Gly-Asp(RGD; cell-adhering peptide)-Alginate scaffolds significantly promotedtarget delivery of cells compared to unmodified gels. By contrast,injection of free cells (bolus) did not promote localization of cells(bioluminescent signal absent).

FIG. 5A is a schematic showing preparation of an autologous (e.g.,syngeneic) alginate-based active cryogel vaccine containing livingattenuated B16-F10 melanoma cells or living attenuatedHER-2/neu-overexpressing breast cancer cells for the prophylactic andtherapeutic treatments of skin cancer or invasive mammary carcinoma,respectively, in mice (e.g., C57BL/6 mice). CpG-ODN (TLR9-based immuneadjuvant) & GM-CSF (cytokine with adjuvant benefits) loaded RGD-modifiedalginate cryogels prepared by a cryogelation process at subzerotemperature were seeded with irradiated B16-F10 cells or irradiatedHER-2/neu-overexpressing breast cancer cells and incubated for 6 h priorto animal vaccination via subcutaneous injection. FIG. 5B is a scanningelectron microscopy (SEM) image showing homogeneous macroporousmicrostructure throughout the square-shaped sponge-like gel construct.FIG. 5C is a SEM cross-sectional image of alginate cryogel showinginterconnected macroporous network. FIG. 5D is a 2-D confocal micrographdisplaying immobilization of irradiated B16-F10 cells on a typicalRGD-containing cryogel after 6 h culture. FIG. 5E is a 3-D reconstructedconfocal fluorescence micrograph of irradiated B16-F10 cells depictingcell-substratum adhesion, spreading, and elongation after 6 h culture ina cryogel vaccine.

FIG. 6 is a bar graph showing immunity against B16F10 challenge inducedby different vaccination protocols. Infection-mimicking microenvironmentfrom injectable alginate-based cryogel conferred potent anti-tumorimmunity. A comparison of the survival time in mice treated withCryogels; (C) antigen+GM-CSF+CpG-ODN (0.2×10⁶ irradiated B16F10 melanomacells+3 μg GM 100 μg CpG), antigen+GM-CSF (0.4×10⁶-CSF+(D) 6 irradiatedB16F10 melanoma cells+3 μg GM), (E) antigen+CpG-ODN (0.4×10⁶ irradiatedB16F10 melanoma cells+100 μg CpG). Animals were also immunized using0.4×10⁶ B16F10 melanoma cells transduced with the murine GM-CSF gene (A)and bolus injections of 0.4×10⁶ irradiated B16F10 melanoma cells+3 μgGM-CSF+100 μg CpG-ODN (B). Mice were challenged (Day 6) with 10⁵ B16-F10melanoma tumor cells and monitored for the onset of tumor occurrence.Each group contained 10 mice.

FIG. 7A is a line graph showing the degree of cell recruitment andexpansion at the injection site and at secondary lymphoid organs (lymphnodes (LNs) and spleen) in response to cryogel vaccination andchallenge. The in vivo proliferative responsiveness of the cells wasassessed by cell counting. The inset shows the chronological order ofthe immunization and tumor challenge. FIG. 7B is a line graph showingthat local delivery of cryogel vaccines promotes recruitment of CD11c(+)and proliferation of CD3(+) T cells. Cryogel vaccines co-deliveringGM-CSF (1.5 μg), CpG-ODN (50 μg), and presenting attenuated B16F10melanoma cells stimulate potent local and systemic CD11c(+) DC andCD3(+) T cells in secondary lymphoid organs (LNs and spleen) as well asin the cryogel scaffolds. C, V, and VC groups correspond to miceinjected with blank cryogels at day 0 (C), mice immunized with cryogelvaccines at day 0 (V), and mice immunized with cryogel vaccines at day0+tumor challenged at day 6 (VC), respectively. Values in (FIGS. 7A-7B)represent mean and standard deviation (n=5).

FIG. 8 is a line graph showing controlled release of GM-CSF for DCrecruitment and programming. Cumulative release of GM-CSF fromAlginate-based cryogel matrices over a period of 2 weeks; (A) 3 μgGM-CSF, (B) 3 μg GM-CSF+100 μg CpG-ODN, (C) PLG microsphere containing 3μg GM-CSF. Values represent mean and standard deviation (n=5).

FIG. 9 is a line graph showing cryogel-enhanced plasmid DNAtransfection. Relative bioluminescence over time for cells transfectedwith a luciferase expression plasmid (150 μg/cryogel, 2injections/animal). Cryogels assist in efficient delivery and celltransfection of polyethylenimine (PEI)/plasmid DNA (blue) when comparedto naked PEI/DNA (red). Values represent mean and standard deviation(n=5). The inset is a photograph that shows a representative localizedlight emission in response to application of firefly luciferin after 29d post injection in mice inoculated with PEI/DNA-containing cryogels.

FIG. 10 is a nuclear magnetic resonance (NMR) spectrum showing the ¹HNMR spectrum of MA-alginate with its characteristic vinylic peaks(˜δ5.3-5.8 ppm). Deuterated water (D₂O) was used as solvent, and thepolymer concentration was 1% (wt/vol). The efficiency of alginatemethacrylation was calculated based on the ratio of the integrals foralginate protons to the methylene protons of methacrylate. MA-alginatemacromonomer was found to have approximately a degree of methacrylation(DM) of 50%.

FIG. 11 is a set of two line graphs showing ¹H NMR of uncross-linked(left) and cryopolymerized (right) 1% wt/vol MA-alginate in D₂O.Cryogelation is induced directly in an NMR tube. 1 mL of macromonomersolution containing the initiator system was transferred into the NMRtube before cryogenic treatment at −20° C. for 17 hr. The vinylic peaks(between 65.3-5.8 ppm) disappeared after cryo-crosslinking. Theconversion was evaluated by comparing the relative peaks ofuncross-linked and cross-linked methylene protons.

FIG. 12 is a series of four photographs showing scanning electronmicroscopic images of free PLGA microspheres (top left) and PLGAmicrospheres dispersed in a alginate square-shaped cryogel (top rightand bottom).

FIG. 13 is a series of two photographs showing that cells injected viathe cryogels have a low apoptosis and cell death. In this example, aRGD-containing peptide was chemically attached to the cryogels toimprove cell adhesion to the 3D-structure alginate-based scaffolds. Cellviability, spreading, and actin cytoskeleton organization process wasassessed by confocal microscopy. Cells colonize the porous structure ofthe alginate-based cryogel and were observed to be growing inside thepores. (Left) live/dead cell viability assay of D1 mesenchymal stemcells (MSC, Id incubation post-injection) and (right) confocal imageshowing injected D1 MSC (6 d incubation post-injection) in RGD-modifiedMA-alginate cryogels.

FIGS. 14A-14B are a set of photographs depicting cryogel (4×4×1 mm)before (FIG. 14A) and after (FIG. 14B) syringe injection in the subcutisof a mouse. These photographs show that the injectable square-shapedalginate sponge-like cryogels have shape-memory properties.

FIGS. 15A-15D are a set of graphs showing the controlled release ofbiologically active immunomodulators from the cryogels. FIG. 15A is abar graph showing the encapsulation efficiency of GM-CSF in alginatecryogels following polymerization, washing, and sterilization. FIG. 15Bis a plot showing the release of GM-CSF for DC recruitment andprogramming. The cumulative release of GM-CSF from alginate cryogelmatrices over a period of 6 weeks is shown. (diamond) 1.5 μg GM-CSF,(circle) 1.5 μg GM-CSF+50 μg CpG-ODN. FIG. 15C is a bar graph showingthe encapsulation efficiency of CpG-ODN in alginate cryogels postpolymerization, washing, and sterilization. FIG. 15D is a plot showingthe release of CpG-ODN for DC activation. The cumulative release ofCpG-ODN from alginate cryogel matrices over a period of 6 weeks isshown. (diamond) 50 μg CpG-ODN, (circle) 1.5 μg GM-CSF+50 μg CpG-ODN.Values represent mean and standard deviation (n=5). Differences betweengroups were statistically significant (* P<0.05, ** P<0.01).

FIGS. 16A-16E are a set of photographs depicting representative swellingat the injection vaccination site. FIG. 16A is a set of two photographsshowing a typical cryogel vaccine before (left) and after (right)seeding with irradiated B16-F10 cells. FIG. 16B is a photograph of mice,where group C mice were vaccinated with cryogel vaccines. Significantswelling was detected at day 13 only for vaccinated with cryogelvaccines (group C). FIG. 16C is a zoomed in photograph of arepresentative mouse from group C, with arrows pointing to sites ofswelling. FIG. 16D is a close-up photograph of swelling in a mousevaccinated with cryogel vaccines. FIG. 16E is another photograph of arepresentative mouse from group C with arrows pointing to sites ofswelling.

FIGS. 17A-17D are a set of schematics and graphs showing the in vitroactivation of differentiated bone marrow derived dendritic cells (BMDC)in response to CpG-ODN-loaded cryogels. BMDC were cultured for 24 h incontact with medium (group A, negative control), blank cryogels/medium(group B), CpG-ODN loaded cryogels/medium (group C), or CpG-ODN/medium(group D, positive control). FIG. 17A is a cartoon depicting the processof bone marrow isolation from murine tibias and femurs, differentiation,and expansion of BMDC to assess their activation in response of releasedCpG-ODN from cryogel vaccines. FIG. 17B is a bar graph showing thefraction of CD11C(+) BMDC used in each condition. FIG. 17C is a bargraph showing the fraction of activated CD86(+) MHCII(+) BMDC in eachcondition. FIG. 17D is a bar graph showing the production of IL-12 inculture media in response to DC stimulated by CpG-ODN in each condition.Values represent mean and standard deviation (n=5). Differences betweengroups were statistically significant (* P<0.05, ** P<0.01, ***P<0.001).

FIG. 18 is a bar graph showing the viability of differentiated BMDCsisolated from murine tibias and femurs used in each of four conditionstested (groups A-D). Differentiated bone marrow-derived dendritic cells(BMDCs) were cultured for 24 h in contact with medium (group A, negativecontrol), blank cryogels/medium (group B), CpG-ODN loadedcryogels/medium (group C), or CpG-ODN/medium (group D, positivecontrol).

FIGS. 19A-19E are a schematic, bar graphs, and images depicting that thecryogel vaccines promote cellular infiltration and leukocytesrecruitment. FIG. 19A is a schematic representation displaying thesubcutaneous injection of cryogel vaccines in mice using a standardgauge needle, as well as local edema and induration at the injectionsite and the recruitment and activation of naïve DC. FIG. 19B is a bargraph showing the enhancement of cellular infiltration in macroporouscryogel sponges versus conventional nanoporous hydrogels. Thedifferences between the macroporous cryogel sponges of the invention andconventional nanoporous hydrogels arise from their methods of formation.The cryogels are formed when the solution is in a partially frozenstate, while the conventional nanoporous gels are not—this results information of large interconnected pores in cryogels, versus nanometerscale pores in conventional gels. These differences in formation lead todifferences in the mechanical properties of these gels, e.g., shapememory.

FIG. 19D is a bar graph showing that GM-CSF delivery from cryogel spongepromotes CD11b(+)CD11c(+) DC recruitment to the cryogel sponges. FIGS.19C and 19E are H&E stainings of sectioned cryogel scaffolds injectedsubcutaneously in the backs of C57BL/6J mice after 1 day: blankscaffolds (C) and GM-CSF (1.5 μg)-loaded scaffolds (E). Values representmean and standard deviation (n=5). Differences between groups werestatistically significant (* P<0.05, ** P<0.01).

FIGS. 20A-20B are a set of flow cytometry plots showing an enhancedrecruitment of CD11b+CD11c+DC from GM-CSF loaded cryogels. Unlike blankcryogels (FIG. 20A, control), FACS analysis for CD11b+CD11c+DC showedthat cryogel vaccines increased the fraction of infiltrated dendriticcells (FIG. 20B, GM-CSF loaded cryogel scaffolds).

FIG. 21 is a set of four photographs showing the magnitude of the immuneresponse elicited by the infection mimic-containing cryogel vaccines. Inparticular, the lymph nodes of vaccinated mice were markedly enlarged.Mice were vaccinated at day 0, and mice were euthanized and spleensexplanted at different time points. Control (naïve mice), Challenged(naïve mice challenged at day 6), Vax (naïve mice vaccinated at day 0),Vax+Challenged (naïve mice vaccinated at day 0 and subsequentlychallenged at day 6).

FIGS. 22A-22G are a set a bar graphs showing that co-delivery ofimmunostimulatory factors (CpG-ODN and GM-CSF) from whole tumorcell-seeded cryogel vaccines stimulate recruitment and activation of DC,amplify CD8(+) cytotoxic T cells, and attenuate FoxP3(+) Treg cells.FIGS. 22A-22C are bar graphs showing the numbers of CD11c(+), pDC, andCD8(+) at day 9 post-immunization isolated from explanted cryogelvaccines (FIG. 22A), LNs (FIG. 22B), and spleen (FIG. 22C). FIGS.22D-22E are bar graphs showing the numbers of CD3(+) T cells (FIG. 22D)and CD8(+) T cells (FIG. 22E) at day 13 post-immunization isolated fromexplanted cryogel vaccines, LNs, and spleen. FIG. 22F is a bar graphshowing the ratio of CD8(+) T cells versus FoxP3(+) Treg cells residingwithin cryogel vaccines, LNs, and at day 23 after immunization. FIG. 22Gis a bar graph showing the in vivo concentrations of mouse cytokines(IL-1α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13,IL-17A, Eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MCP-1α, MCP-1β,RANTES, TNF-α) from explanted cryogels at day 13. C, V, and VC groupscorrespond to mice injected with blank cryogels at day 0 (group C), miceimmunized with cryogel vaccines at day 0 (group V), and mice immunizedwith cryogel vaccines at day 0+tumor challenged at day 6 (group VC),respectively. Values in FIGS. 22A, 22B, 22C, 22D, 22F, and 22G representmean and SD (n=5). *P<0.05, **P<0.01, ***P<0.001 versus all otherexperimental conditions unless otherwise noted.

FIGS. 23A-23D are a set of graphs showing the prophylactic efficacy ofcryogel vaccines and long term protection against melanoma cancer. Theinfection-mimicking cryogel microenvironment confers potent anti-tumourimmunity. FIG. 23A is a Kaplar meier plot showing the comparison of thesurvival rate in challenged C57BL/6 mice treated with (group A): BolusG-Vax injection (irradiated GM-CSF secreting B16-F10 cells; positivecontrol); (group B): Bolus injection (irradiated B16-F10cells+CpG-ODN+GM-CSF); (group C): Cryogel vaccine (irradiated B16-F10cells+CpG-ODN+GM-CSF; vaccine of interest); (group D): Cryogel vaccine(irradiated B16-F10 cells+GM-CSF); (group E): Cryogel vaccine(irradiated B16-F10 cells+CpG-ODN); (group F): Blank cryogel (control;negative control); or (group G): naïve mice (no immunization). At day 6following immunization, C57BL/6J mice (10 mice/group) were challengedwith 10⁵ B16-F10 tumor cells and monitored for animal survival. FIG. 23Bis a tumor growth curve after the 1st tumor challenge (5×10⁵ cells)following one single cryogel vaccination 6 days prior to the challenge.FIG. 23C is a Kaplar meier plot showing a comparison of the survivalrate in re-challenged mice treated with (group A): Bolus G-Vax injection(irradiated GM-CSF secreting B16-F10 cells; positive control); (groupB): Bolus injection (irradiated B16-F10 cells+CpG-ODN+GM-CSF); (groupC): Cryogel vaccine (irradiated B16-F10 cells+CpG-ODN+GM-CSF; vaccine ofinterest); (group D): Cryogel vaccine (irradiated B16-F10 cells+GM-CSF);(group E): Cryogel vaccine (irradiated B16-F10 cells+CpG-ODN); (groupF): Blank cryogel (control; negative control); (group G): naïve mice (noimmunization). At day 126 following immunization, C57BL/6J mice (10mice/group) from the first challenge study were challenged a second timewith 10⁵ B16-F10 tumor cells and monitored for survival. FIG. 23D is abar graph showing the overall survival rate after two consecutivetumor-challenges in mice to evaluate long-term immunological protectionin the context of melanoma. Values represent mean and SD (n=10 percondition).

FIG. 24 is a bar graph showing that vaccination of mice withsyringe-injectable cryogels elicits a strong humoral antitumor immuneresponse against invasive HER-2/neu-overexpressing breast cancer cells.BALB/c mice (n=5) were immunized with (G1) cryogels(CpG-ODN+GM-CSF+breast tumor lysates); (G2) cryogels (CpG-ODN+attenuatedGM-CSF secreting HER-2/neu-overexpressing breast cancer cells); (G3)cryogels (CpG-ODN+GM-CSF)+attenuated HER-2/neu-overexpressing breastcancer cells); (G4) CpG-ODN+attenuated GM-CSF secretingHER-2/neu-overexpressing breast cancer cells; (G5) attenuated GM-CSFsecreting HER-2/neu-overexpressing breast cancer cells; (G6) blankcryogels; (G7) naïve mice; (G8) challenged naïve mice. At day 30following immunization, the mice were challenged subcutaneously with 105HER-2/neu-overexpressing breast tumor cells.

FIG. 25 is a Kaplar meier curve showing survival of mice immunized usingHER-2/neu protein-specific vaccines. Autologous attenuatedHER-2/neu-overexpressing breast cancer cell-based cryogel vaccineconfers potent anti-tumour immunity. A comparison of the survival ratein challenged mice treated with (Group 1): Alginate-based cryogelvaccine (Lysates+CpG-ODN+GM-CSF); (Group 2): Alginate-based cryogelvaccine (GM-CSF secreting cells+CpG-ODN); (Group 3): Alginate-basedcryogel vaccine (cells+CpG-ODN+GM-CSF; vaccine of interest); (Group 4):Bolus G-Vax injection (GM-CSF secreting cells; positive control); (Group5): Bolus injection (GM-CSF secreting cells+CpG); (Group 6): Blankalginate-based cryogel (control; negative control); (Groups G, and E):naïve mice (no immunization). At day 30 following immunization, BALB/cmice (5 mice/group) were challenged with 105 HER-2/neu-overexpressingbreast tumor cells and monitored for animal survival.

FIGS. 26A-26C depict the fabrication of gelatin cryogels with highlyinterconnected pores. FIG. 26A is a schematic of methacrylated gelatin(GelMA) synthesis and crosslinking. Pendant methacrylate groups areadded primarily to the free amines of gelatin by reaction withmethacrylic anhydride. Free radical polymerization results in crosslinkformation between methacrylate groups. FIG. 26B is a schematic showingcryopolymerization of methacrylated gelatin. Freezing of methacrylatedgelatin in the presence of radical initiators (ammonium persulfate (APS)and N,N,N′,N′-tetramethylethylenediamine (TEMED)) allows polymerizationto occur in the partially frozen state (cryopolymerization). Icecrystals formed during the freezing process and thawing aftercryopolymerization results in the formation of a hydrogel withmicron-scale pores. FIG. 26C is a graph showing the % interconnectedporosity of the gelatin cryogel when various concentrations of GelMA areused. Volume of interconnected pores in gelatin cryogels (normalized tototal gel volume). Values represent mean and standard deviation (n=10).Data were compared using Analysis of variance (ANOVA) with Bonferroni'spost-hoc test (**p<0.01, ***p<0.001).

FIG. 27 is a pair of nuclear magnetic resonance (NMR) spectra comparingcontrol gelatin (top) and methacrylate modified gelatin (bottom). ¹H-NMRspectra of unmodified (top) and methacrylated (bottom) gelatin shows theappearance of the vinylene protons (red box) of the methacrylate pendantgroup after reaction of gelatin with methacrylic anhydride.

FIGS. 28A-28C depict mechanical properties of gelatin cryogels. Hydrated1.5% and 2% cryogels were tested in compression at 1 mm/min. Theresulting stress-strain curves were used to calculate the (FIG. 28A)elastic modulus, (FIG. 28B) fracture stress, and (FIG. 28C) fracturestrain. FIG. 28A is a graph depicting the elastic modulus. FIG. 28B is agraph depicting the fracture stress. FIG. 28C is a graph depicting the %fracture strain. Error bars represent the standard deviation frommeasurements of 10 gels tested per condition. Data were compared using atwo-tailed unpaired Student's t-test with Welch's correction (**p<0.01,****p<0.0001). The mechanical properties of 1.0% cryogels could not bemeasured using compressive mechanical testing.

FIGS. 29A-29B are a series of images showing the microarchitecture ofgelatin cryogels. FIG. 29A is a set of images showing the surface andcross-sectional scanning electron microscopy (SEM) micrographs of highlyporous 1.0% (w/v) gelatin cryogels (scale bar=50 am). FIG. 29B is animage using 2-photon imaging at a depth of 150 am below the surface of arhodamine-gelatin cryogel (scale bar=1 mm). The inset shows a magnifiedview at the center of the scaffold diameter (scale bar=100 μm). Imagesare representative of at least 5 gels imaged using each modality.

FIGS. 30A-30C are a series of images depicting bulk mechanical behaviorand injectability of gelatin cryogels. FIG. 30A is a panel of imagesdemonstrating the ability of an individual rhodamine-gelatin cryogel tobe compressed between forceps (dashed white line) to large strain,followed by release and resumption of its original shape. FIG. 30B is apanel of images showing Rhodamine-gelatin cryogel following collapse dueto wicking of free water (0 ms), and rapid rehydration followingexposure to excess DPBS. FIG. 30C is a panel of images showingRhodamine-gelatin cryogel exiting the bore of a 16 G needle (0 ms, outerneedle wall outlined with dashed line), and its expansion to originalsize and shape after injection. All scale bars=2 mm.

FIGS. 31A-31E depict cell attachment, survival, and proliferation ongelatin cryogels in vitro. FIG. 31A is a representative 2-photonmicroscopy image of 5-Chloromethylfluorescein Diacetate (CMFDA)-labeledcells at 150 μm depth below the surface of a rhodamine-gelatin cryogel 2h after seeding (n=5, scale bar=200 μm). FIG. 31B is a SEM micrograph of3T3 cells spread on cryogel surface at 1 d post-seeding (n=5, scalebar=10 μm). Cells are false-colored for emphasis. FIG. 31C is a stainingof F-actin on histological sections showing cell spreading withinscaffolds after one day of culture (n=3, scale bar=25 μm). FIG. 31D is agraph showing an analysis of the number of live cells, overall metabolicactivity as measured by alamarBlue reduction, and viability of cellsrecovered from gelatin cryogels over time in culture. Values representthe mean and standard deviation (n=3). FIG. 31E is a staining for newDNA synthesis within the scaffold using 5-ethynyl-2′-deoxyuridine (EdU)incorporation (n=3, scale bar=100 am).

FIGS. 32A-32B are a set of SEM images of cryoGelMA gels three days after3T3 cell seeding. FIG. 32A is a SEM image showing a monolayer of 3T3cells after three days of culture on cryoGelMA. FIG. 32B is a SEM imageshowing extracellular matrix deposition on the cryoGelMA surface seenafter mechanically disrupting the cell monolayer.

FIGS. 33A-33B are live dead imaging of 3T3 cells following seeding ongelatin cryogels. Gelatin cryogels were labeled with a Live/Deadstaining kit (green=live, red=dead) to assess viability. FIG. 33A is abulk image at 1 day after seeding. FIG. 33B is a bulk images at 3 daysafter seeding. Magnified image of the cellular coverage of the cryogelsurface over the culture period (insets). Scale bar=1 mm, inset scalebar=200 μm.

FIG. 34 is a graph showing bromodeoxyuridine (BrDU) incorporation into3T3 cells. 3T3 cells seeded on tissue culture polystyrene (˜50%confluency) and for one day on cryoGelMA (n=3) were pulsed with 30 mBrDU for 4 h and analyzed by flow cytometry for BrDU incorporation.Values represent percentage of total cells that exhibited positive BrDUstaining. Data were compared using a two-tailed unpaired Student'st-test with Welch's correction (***p<0.001).

FIGS. 35A-35C depict enzymatic degradation of gelatin cryogels in vitro.FIG. 35A is a graph showing in vitro degradation of rhodamine-gelatincryogels (n=3) in the presence of 25 U/ml collagenase type II. FIG. 35Bshows Zymography with mouse and human pro-MMP-2 and pro-MMP-9 usingGelMA-polyacrylamide gels. FIG. 35C is a graph showing thequantification of degradation of rhodamine-gelatin cryogels (n=4) in thepresence of 10 jag/ml recombinant mouse and human MMP-2 and -9 for 18hours. One-way ANOVA with Dunnett's test was performed to compare allMMP-treated conditions with the control gels incubated in buffer alone(n=4, ****p<0.0001). Values represent the mean and standard deviation inall plots.

FIGS. 36A-36D are a set of images depicting in vivo injection of gelatincryogels. FIG. 36A is an in vivo fluorescence image of rhodamine-gelatincryogel in a C57Bl/6J mouse immediately following subcutaneousinjection. FIG. 36B is an in situ image of rhodamine-gelatin cryogelunder the skin at 1 w and 2 m following implant. FIG. 36C is anHematoxylin and eosin (H&E) stain at 1 w following implant at thecryogel-tissue interface (left) and the cryogel interior (right) (n=3,scale bar=50 jam). Arrows indicate the cryogel-host border. FIG. 36D isan H&E stain at 2 m following implant at the cryogel-tissue interface(left) and the cryogel interior (right) (n=3, scale bar=50 jam). Arrowsindicate the cryogel-host border.

FIGS. 37A-37E depicts in vivo cell recruitment to gelatin cryogels bysustained release of GM-CSF. FIG. 37A is a schematic of cell recruitmentto GM-CSF-releasing gelatin cryogels. Sustained release of GM-CSF fromthe cryogel implant creates a chemoattractant gradient to attract hostimmune cells. FIG. 37B is a graph showing in vitro cumulative GM-CSFrelease from gelatin cryogels. FIG. 37C is a graph showing the averagerelease rate of GM-CSF from gelatin cryogels. FIG. 37D is a graphshowing recruited cell numbers in blank and GM-CSF-releasing gelatincryogels at 14 d post-implant (Student's t-test, n=3 mice, **p<0.01).FIG. 37E is a set of representative H&E stainings from blank andGM-CSF-releasing cryogels 14 d after implantation in C57/B16J mice (n=3,scale bar=500 jam). Inset shows a magnified view of the scaffoldinterior (scale bar=20 μm). Arrows indicate the cryogel-tissue borders.Values represent the mean and standard deviation in all plots.

FIGS. 38A-38E depict gelatin cryogel degradation by recruited cells invivo. FIG. 38A is a set of images from longitudinal in vivo imaging ofthe degradation of blank and GM-CSF-releasing rhodamine-gelatin cryogelsin C57Bl/6J-Tyr^(c-2J) mice (n=5). FIG. 38B is a graph showing thequantification of fluorescence signal from longitudinal in vivo imagingof the degradation of blank and GM-CSF-releasing rhodamine-gelatincryogels in C57Bl/6J-Tyr^(c-2J) mice (n=5). FIG. 38C is an image showinggelatin zymography of cellular lysates from 1 w implanted blank andGM-CSF-releasing rhodamine-gelatin cryogels (n=3). FIG. 38D is arepresentative in vivo fluorescence image of MMPSense 750 FASTactivation in mice 7 d following injection in opposite flanks with blankand GM-CSF-releasing rhodamine-gelatin cryogels. Scaffold borders areoutlined in white. FIG. 38E is a graph depicting quantitation (pairedt-test, n=3, **p<0.01) of MMPSense 750 FAST activation in mice 7 dfollowing injection in opposite flanks with blank and GM-CSF-releasingrhodamine-gelatin cryogels. Scaffold borders are outlined in white.Values represent the mean and standard deviation in all plots.

FIGS. 39A-39C are images of cryoGelMA gels implanted for 17 days. FIG.39A is an image of a blank cryoGelMA gel. FIG. 39B is an image of aGM-CSF releasing cryoGelMA gel. FIG. 39C depicts the sizes of recoveredblank versus GM-CSF releasing cryoGelMA gels after injection into mice.

FIG. 40A is a histological image of the center of a blank cryoGelMAscaffold 14 days after subcutaneous injection into mice. FIG. 40B is ahistological image of the center of a GM-CSF releasing cryoGelMAscaffold 14 days after subcutaneous injection in mice.

FIG. 41 is a histological image of the injection site of GM-CSFreleasing cryoGelMA in a mouse, depicting the presence of macrophages.

FIGS. 42A-42F are a set of graphs indicating the number of cells ofvarious types recruited to the cryoGelMA at various doses of GM-CSF.Error bars represent the standard deviation of the mean. Data werecompared using ANOVA with Bonferroni's post-hoc test (*p<0.05). FIG. 42Ashows the number of live cells recruited to the cryoGelMA. FIG. 42Bshows the percent of cells that are macrophages (CD11b+F4/80+). FIG. 42Cshows the percent of cells that are CD4+. FIG. 42D shows the percent ofcells that are granulocytes (CD11b+Gr-1+). FIG. 42E shows the percent ofcells that are dendritic cells/macrophages (CD11b+CD11c+). FIG. 42Fshows the percent of cells that are CD4+ regulatory T cells(CD4+/(CD25+FoxP3+).

FIGS. 43A-43B are a set of graphs showing the proportion of various celltypes as a percentage of all live cells resident in cryoGelMA gels onday 6 and 15 after injection into mice. Error bars represent thestandard deviation of the mean. Data were compared using a two-tailedunpaired Student's t-test with Welch's correction (**p<0.01,***p<0.001). FIG. 43A shows the percentage of cells that areCD11b+CD11c+. FIG. 43B shows the percentage of cells that are NK1.1+.

FIGS. 44A-44B are schematics of various CpG incorporation strategiesinto cryoGelMA gels.

FIG. 45 is a schematic of a CpG incorporation strategy into cryoGelMAgels.

FIG. 46 is a graph showing the rate of CpG release from variouscryoGelMA gels.

FIG. 47A is a bar graph showing the percentage of mouse bone marrowderived dendritic cells that are CD40+ after culturing in cryoGelMAconditioned media. FIG. 47B is a bar graph showing the percentage ofmouse bone marrow derived dendritic cells that are CD86+ after culturingin cryoGelMA conditioned media.

FIG. 48A is a graph showing the percentage of IL-12 positive dendriticcells after culturing in various conditions. FIG. 48B is a graph showingthe percentage of TNF positive cells after culturing in variousconditions. FIG. 48C is a graph showing the percentage of IL-6 positivecells after culturing in various conditions.

FIG. 49 is a fluorescence microscopy image of a histological sectionfrom a cryoGelMA gel after seeding with mouse bone marrow deriveddendritic cells. Sections were stained for F actin (green) and nuclei(blue) 48 hours after seeding.

DETAILED DESCRIPTION

A major drawback in today's surgical implantation of three dimensionalscaffolds is the trauma created by physicians while administering thescaffolds/devices. The compositions and methods described herein reducethe cost and invasiveness of the cell therapy and/or tissue engineeringapproach. Prior to the invention described herein, tissue engineeringand cell therapy often used devices and polymer scaffolds that requiredsurgical implantation. Implantation of polymer scaffolds at a surgicalsite requires anesthesia and incisions, each of which treatment methodshave undesirable side effects. Described herein are compositions andmethods that allow tissue engineers, physicians, and surgeons to engagein cell therapy and tissue engineering applications in a less invasivemanner, thereby removing the need for surgical implantation. Asdescribed in detail below, injectable gel devices were developed toreduce the invasiveness of a cell therapy or tissue engineering system,thereby eliminating the need for, or reduce the size of, any incisionsrequired to implant the material. For a system to be injectable, it mustbe capable of flowing through a hollow small-bore needle. Methods ofimplantation of a gel device or injection of a liquid for polymerizationin situ presented a number of challenges including short response time,proper gelation conditions, appropriate mechanical strength andpersistence time, biocompatibility, and the likelihood to protectprotein drugs or cells in some adverse environments. In order toovercome these limitations, deformable fully-crosslinked and pre-shapedporous gel devices that are easily prepared, processed, and injectedthrough the needle of a syringe was developed.

Earlier injectable hydrogels (e.g., U.S. Pat. No. 6,129,761) allowed forthe formation of scaffolds in situ but had several major drawbacks.First, potential problems occur with in situ polymerization includingheat generation and un-reacted toxic chemicals. Additionally, slowgelation kinetics and in vivo biofluid dynamics involve dispersion ofpre-gel solution leading to poor cell entrapment and physical integrityof the gel. Finally, nanosized pore architecture of scaffolds impedesefficient oxygen delivery, nutrient exchange, cell-movement, andlong-term survivability of tissue cells.

The invention described herein provides a minimally-invasive method ofinjecting preformed macroporous hydrogels that are loaded with cellsand/or therapeutics. Cells are optionally implanted and cultured ontothe polymeric matrix before or after administration to a subject. Foodand Drug Administration (FDA)-approved polymer-based scaffolds thatsupport the attachment and proliferation of cells, degradable andcapable of releasing drugs (e.g., proteins) at a controlled rate in vivoare designed in any desirable size and shape, and injected in situ as asafe, preformed, fully characterized, and sterile controlled deliverydevice. Described in detail below are biologically active cell-seededinjectable scaffolds with structural integrity within the body thatcontrollably deliver growth factors while providing cellular buildingblocks to enhance tissue formation. Seeding and organizing cells priorto administration of macroscopic injectable matrices enhance in vivocell engraftment and provide cell support and guidance in the initialtissue formation stage. This invention is useful for clinicalapplications including artificial extracellular matrix for tissueengineering, dermal filler in cosmetic surgery, controlled releasereservoir for drug and cell delivery, and immune cell reprogramming forcancer vaccines. Additional benefits include less injection pain, lessbleeding/bruising and higher levels of patient satisfaction.

The present invention describes a non invasive strategy to administerlarge-size macroporous biodegradable hydrogels as a 3-D scaffold and adrug delivery platform. Any biocompatible polymers or monomersundergoing cryopolymerization are utilized. Suitable polymers andmonomers include naturally derived polymers (alginate, hyaluronic acid,heparin, gelatin, carob gum, collagen, etc.) and synthetic polymers(poly(ethylene glycol) (PEG), PEGylated glutaminase (PEG-PGA),PEG-poly(L-lactide; PLA), poly(2-hydroxyethyl methacrylate) (pHEMA),PAAm, poly(N-isopropylacrylamide) (PNIPAAm), etc.). This ability to usedifferent materials is useful in different applications and adds afurther degree of versatility to the compositions and methods describedherein. The highly elastic macroscopic scaffolds with spongy-likemorphology are prepared by cryogelation, a technique used to producepolymeric materials with large interconnected pores, high volumefraction porosity within soft, mechanically stable and high waterabsorbing capacity. As described below, the cryogels allow for theinjectability of preformed large-size scaffolds through a needle withoutthe need of an invasive implantation. Flowable material can fill anydefect due to the sponginess of the network. Elastic deformation ofcryogels by external forces (mechanical deformation) led to abrupt gelshrinkage with full shape recovery capability, which is useful in thedesign of injectable preformed scaffolds for cell delivery in aminimally-invasive fashion for tissue engineering and regenerativemedicine.

The use of large-size preformed scaffolds (>1 mm) mimicking theextracellular matrix was evaluated. Described herein is the design oflarge biomaterials with various shapes and sizes ranging from 2 mm up to8 mm that are employed as injectable cell-laden scaffold cryogels.Injectable macroscopic hydrogels are supplied in individual treatmentsyringes for single patient use and ready for injection (implantation).The gel, consisting of crosslinked alginate suspended in a physiologicbuffer, is a sterile, biodegradable, non-pyrogenic, elastic, clear,colorless, homogenized scaffold implant. The injectable gels arepackaged in proprietary luer-lock syringes that are injected via a16-gauge or smaller diameter needle depending on the size of the gel.

The strategies described herein are for delivery of preformedbiomaterials suitable for minimally invasive therapies. Injectablemacroscopic biomaterials are useful as surgical tissue adhesives,space-filling injectable materials for hard and soft tissue repair, drugdelivery, and tissue engineering. Described herein is an approach ofpure alginate scaffolds fabrication, which resulted in the formation of,interconnected, superporous network (pore size in the range of 10 μm-600μm). These spongy-like gels are highly flexible and squeezable, capableof releasing up to 70% of their water content without altering the gelmicrostructure. Optionally, the gel further includes a large range ofpurified polymers such as hyaluronic acid, heparin, carob gum, gelatinetc; or a cell adhesive molecule such as fibronectin, or integrinbinding peptide. In addition, the hydrogel is used as a drug reservoirfor the controlled delivery of one or more therapeutic agents.Alginate-based gels have excellent mechanical properties, elongation,and fast shape recovery by elasticity. The shape of the gels, which wasdeformed by an external force (e.g., shear stress), was recovered byswelling in a very short time (<1 s). This recovery had good persistenceand repeatability. The superporous (e.g., greater than 75% porosity)scaffolds described herein offer significant advantages such asinjectability and easy and efficient cell encapsulationpost-polymerization. For example, the cryogels are characterized byporosities of 80-90% or more. Animal studies were performed to examinethe integration of the spongy-like gels with the host tissue show thatthe alginate-based scaffolds are biocompatible and do not elicit animmune response or rejection when injected in mice.

Over the past few decades, whole cell autologous cancer vaccines havebeen used for broad, tumor associated antigen presentation andpatient-specific cancer immunotherapy. However, only a limited clinicalefficacy has been achieved. To improve cell-based cancer vaccinesefficacy, the invention features a vaccination system enablingrecruitment of dendritic cells while creating an immunogenicmicroenvironment consisting of irradiated tumor cells along withadjuvant could enhance T-effector responses and therefore protectiveimmunization. To this end, a sponge-like macroporous biomaterial systemwas prepared by cryogelation and designed to not only to improve tumorcell engraftment, but also to control the release of immunomodulatorssuch as GM-CSF (recruitment factor) and CpG (programming factor), andfinally to enhance immune-cell trafficking and activation in situ.Unlike most surgically implantable scaffolds, the injectablecryogel-based vaccine is minimally invasive and can be administratedthrough a conventional small-bore needle. Upon subcutaneous injection ofthe vaccine depot into mice, T effector responses were boosted,resulting in protective immunity in the context of melanoma. Protective,safe, and long-term anti-tumor immunity was generated with thesematerials, as 80% survival was achieved in animals that otherwise diefrom cancer within 30 days. As described in detail in the Examplesbelow, 100% survival was achieved after tumor rechallenge on vaccinatedmice following 4 months post vaccination, demonstrating a specific,strong, and durable immunological protection in the event of tumorrecurrence. This injectable material-based vaccination regime holds highpromises to replace traditional whole cell vaccination.

Synthesis of Methacrylated-Alginate (MA-Alginate) and Other ModifiedPolymers

Methacrylated alginate (MA-alginate) was prepared by reacting highmolecular weight alginate with aminoethyl methacrylate (AEMA). Tosynthesize methacrylated alginate with 100% theoretical methacrylationof uronic acid carboxylate groups, high molecular weight sodium alginate(1 g) was dissolved in a buffer solution (0.6% w/v, pH ˜6.5) of 100 mMMES containing 0.5 M NaCl. N-Hydroxysuccinimide (NHS, 1.3 g) andN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 2.8g) was added to the reaction mixture to activate the carboxylic acidgroups of the alginate. After 5 min, AEMA (2.24 g, molar ratio ofNHS:EDC:AEMA=1:1.3:1.1) was added to the product and the reaction wasmaintained at room temperature for 24 h. The mixture was precipitatedwith the addition of excess of acetone, filtered, and dried in a vacuumoven overnight at room temperature. ¹H NMR was used to confirm thechemical modification of alginate and characterize the degree offunctionalization of MA-alginate (FIG. 10).

Any biocompatible water-soluble polymer or monomer can be used to makeinjectable cryogels. Several monomers/polymers or a combination ofpolymers have been used to make the injectable cryogel devices describedherein, e.g., hyaluronic acid, gelatin, heparin, dextran, carob gum,PEG, PEG derivatives including PEG-co-PGA and PEG-peptide conjugates.For example, the polymers may be a combination of degradable andnon-degradable synthetic polymers and natural polymers (polysaccharides,peptides, proteins, DNA). Biocompatible synthetic polymers includePolyethylene glycol (PEG), Polyvinyl alcohol (PVA), Poly(2-hydroxyethylmethacrylate) (PHEMA), Poly(N-isopropylacrylamide) (PNIPAAm),Poly(acrylic acid) (PAAc), Polyesters (e.g. Polylactide, Polyglycolide,Polycaprolactone), and Polyanhydrides. Naturally-occurring polymersinclude Carbohydrates (e.g. Starch, Cellulose, Dextrose, Alginate,Hyaluronic Acid, Heparin, Dextran, Gellan Gum, etc), Proteins (e.g.Gelatin, Albumin, Collagen), Peptides, and DNA. All compositions arepurified prior to fabrication of the hydrogels.

In addition to the free radical polymerization process to cross-link thepolymers and make chemically cross-linked injectable cryogels(polymerization time is about 17 hr), gels are optionally polymerizedusing other processes. Injectable cryogels can be classified under twomain groups according to the nature if their cross-linking mechanism,namely chemically and physically cross-linked gels. Covalentcross-linking processes include radical polymerization (vinyl-vinylcoupling), michael-type addition reaction (vinyl-thiol cross-linking),Condensation (carboxylic acid-alcholol and carboxylic acid-aminecross-linking), Oxidation (thiol-thiol cross-linking), Click chemistry(1,3-dipolar cycloaddition of organic azides and alkynes), Diels-Alderreaction (cycloaddition of dienes and dienophiles), Oxime, Imine andHydrazone chemistries. Non-covalent cross-linking include Ioniccross-linking (e.g. calcium-crosslinked alginate), Self assembly (phasetransition in response to external stimuli, such as Temperature, pH, ionconcentration, hydrophobic interactions, light, metabolite, and electriccurrent).

Cryogel Fabrication

Cryogel matrices were synthesised by redox-induced free radicalpolymerization of, e.g., MA-alginate in water. Alginate cryogels aresynthesized by mixing 10 mg (1% wt/v) of MA-alginate macromonomer indeionized water with TEMED (0.5% wt/v) and APS (0.25% wt/v). The mixtureis immediately poured into a pre-cooled Teflon mold and frozen at −20°C. After cryo-crosslinking has finished, gels are heated to roomtemperature to remove ice crystals, and washed with distilled water.Cell-adhesive cryogels were synthesized using a RGD-containing peptidecomposition, e.g., ACRL-PEG-G4RGDASSKY (SEQ ID NO: 2) as a comonomer(0.8% wt/v) during the polymerization. (Acryloyl is abbreviated ACRL.)By mixing the RGD-containing peptide composition (monomers) with thealginate, the RGD becomes chemically attached (covalently attached) tothe polymer structure. RGD integrin-binding motif was used to promotecell-substrate interactions. NMR spectroscopy was used to characterizevinyl conversion of MA-alginate macromonomer after cryopolymerization.As shown in FIG. 2, full disappearance of methylene protons (between5.3-5.8 ppm) for MA-alginate macromonomer (1% wt/v) was reached afterthe cryopolymerization process in the presence of the initiator system(APS/TEMED). This indicates that high vinyl conversions can be achievedfor cryogels (see FIG. 11). Injectable cryogels can be prepared atdifferent concentrations depending on the MW and the degree of chemicalmodification of the polymer itself (1% wt/v was chosen as a proof ofconcept).

As described above, RGD remains attached to the polymer structure byvirtue of covalent bonding (co-polymerization). However, certainbiomolecules are to be released following administration of the cryogelto the subject. In this case, the biomolecules are simply mixed with thepolymer prior to the cryogelation process.

Cryogelation

Cryogels are a class of materials with a highly porous interconnectedstructure that are produced using a cryotropic gelation (orcryogelation) technique. Cryogelation is a technique in which thepolymerization-crosslinking reactions are conducted in quasi-frozenreaction solution. During freezing of the macromoner (MA-alginate)solution, the macromonomers and initiator system (APS/TEMED) expelledfrom the ice concentrate within the channels between the ice crystals,so that the reactions only take place in these unfrozen liquid channels.After polymerization and, after melting of ice, a porous material isproduced whose microstructure is a negative replica of the ice formed.Ice crystals act as porogens. Pore size is tuned by altering thetemperature of the cryogelation process. For example, the cryogelationprocess is typically carried out by quickly freezing the solution at−20° C. Lowering the temperature to, e.g., −80° C., would result in moreice crystals and lead to smaller pores.

The advantage of these so-called “cryogels” compared to conventionalmacroporous hydrogels obtained by phase separation is their highmechanical stability. They are very tough, and can withstand high levelsof deformations, such as elongation and torsion; they can also besqueezed under mechanical force to drain out their solvent content. Theimproved mechanical properties of alginate cryogels originate from thehigh crosslinking density (highly methacrylated alginate polymerizesinto cross-linked polymer structures with a relatively high crosslinkdensity) of the unfrozen liquid channels of the reaction system. Thus,after polymerization, the gel channels with high polymer content areperfect materials for building the pore walls.

Biomolecules, e.g., GM-CSF, CpG nucleic acids, are entrapped in thepolymer structure but not chemically linked to it. Thus, these moleculesare released from the cryogel by diffusion or gel degradation over time.For example, low molecular weight compositions (less than 10 kDamolecular mass), e.g., CpG oligonucleotides, are released by diffusion.Larger entrapped molecules (greater than about 10 kDa, e.g., 10-50 kDain molecular mass), e.g., proteins, large DNAs, e.g., plasmid DNA, arereleased primarily by cryogel degradation. Human Recombinant GM-CSF(e.g., available from PeproTech, Catalog #300-03) is encoded by thefollowing polypeptide sequence (SEQ ID NO:5):

MAPARSPSPS TQPWEHVNAI QEARRLLNLS RDTAAEMNET VEVISEMFDL QEPTCLQTRLELYKQGLRGS LTKLKGPLTM MASHYKQHCP PTPETSCATQ IITFESFKEN LKDFLLVIPFDCWEPVQE Injectable Hybrid Cryogels

Injectable delivery systems for therapeutic proteins (e.g., hydrogelsand microspheres) have attracted wide attention. Conventional hydrogels,however, typically release their hydrophilic contents too rapidly in alarge initial burst, and phagocytes may clear microspheres within arelatively short time period after administration.

Microsphere/cryogel combination systems achieve a controlled andsustained release of proteins as an injectable delivery system. PLGAmicrospheres (size ˜10-50 m) containing a model protein (GM-CSF) wereprepared and then mixed with a MA-alginate pre-gel solution priorcryopolymerization. The mixing ratio of the components was optimized toretain injectability and shape memory properties of pure alginatecryogels. As shown in FIG. 12, PLGA microspheres were physicallyentrapped within the cryogel network (polymeric walls) of cryogels.Also, hybrid cryogel have been created as a carrier for controlleddelivery of hydrophobic and/or low molecule weight drugs. The resultsnot only provide a strategy for delivery drugs from an injectable 3-Dpreformed macroporous scaffolds as a sustained-release drug carrier butalso open an avenue for the design of the hybrid injectable hydrogels.

Other examples of hybrid polymer combinations include cryo-ferrogels andpolydiacetylene-based cryogels. One class of injectable porousbiomaterials for on-demand drug and cell delivery comprisescryo-ferrogels. The magnetic-sensitive scaffolds based on macroporouselastic alginate-based cryo-ferrogels, were fabricated with 3-Dconnected macropores and coupled with magnetic particles (Fe₃O₄ nano-and micro-particles) and cell-binding moieties. Under applied magneticfields, the loaded macroporous ferrogel with biological agents lead tolarge and prompt deformation triggering release of drugs and cells in acontrolled fashion. In another example, injectable color-changingbiomaterials such as polydiacetylene-based cryogels, which change inresponse to external stimuli such as mechanical forces. The materialscontain mechanophore-molecules (e.g., Polydiacetylene Liposome) thatundergo a geometric distortion when a certain amount of force is exertedupon it, leading to a color transition. Smart polymers that change colorwhen the material becomes overstressed are very useful to identifycell-substrate interactions and to accurately measure deformations.

Administration of Injectable Cryogels

Syringes and needles are typically used to introducing the cryogels intothe body. The term “syringe” technically refers to the reservoir (thatholds the liquid) and the plunger (which pushes the liquid out of thereservoir). The “needle” is the part that enters the body, e.g., into avein, under the skin, or into muscle or other tissue. The word “syringe”is also sometimes used to refer to the entire reservoir/plunger/needlecombination. They come in a variety of sizes, e.g., a common reservoirsize is 1 cc (1 cubic centimeter (cc)=1 milliliter), with a 25 gaugeneedle size or smaller.

The needle gauge refers to the size of the bore or hole in the needle.The higher the gauge, the thinner the needle (and the smaller the hole).A 28 gauge needle (abbreviated 28 G) is therefore thinner than a 25gauge needle, which is in turn thinner than an 18 gauge needle. Insulinneedles are typically ½ inch in length and tuberculin needles aretypically 5/8 of an inch in length. As inscribed on packaging, needlelength appears after the gauge number: “28 G 1/2” refers to a 28 gaugeneedle that is ½ inch long.

Larger gauge (frequently 23 G or 21 G), longer needles are often usedfor intramuscular injections. Muscle syringes are typically 1 cc involumes, but larger volumes are sometimes, e.g., 2 to 5 ccs syringes,depending on the application. Larger volumes and larger bores areappropriate for delivery of cryogels for larger scale muscle repair orregeneration, e.g., after extensive or traumatic laceration of tissuesuch as injuries incurred in battle or car/plane accidents. Intravenousinjectors or needles are used for fine or delicate tissue therapy, e.g.,cosmetic dermal filler administration. Such applications typically useshorter needles no larger than 25 G.

Survivability of Cells after Injection

Reversible compactible behavior enables pre-formed cryogels with desiredphysical properties, as characterized ex-vivo, to be delivered in-vivovia application of a moderate non-destructive shear stress duringinjection through a syringe. Studies were carried out to evaluatewhether the fluid velocity, dynamic pressure, and shear stress resultingfrom the injection affects cell viability.

The data indicated that, during the injection, cells integrated in theRGD-modified cryogel were protected by the scaffold from mechanicaldamage. Although adherent cells may experience some shear stress appliedduring the injection, cryogels are capable of absorbing most of theenergy when the scaffolds are compressed, thereby, maintaining high cellviability (92%) and their proliferative potential as shown in FIG. 13.

Thus, the shear stress (or compression) applied to cells in the cryogelas they pass through the bore of a needle or other delivery apparatussuch as a catheter does not measurably hurt or damage the cells withinthe cryogel. Following passage through a needle or other deliveryapparatus, cell viability was routinely 90% or greater.

Cancer Vaccines and Melanoma

Vaccination has been one of the most effective means to increase globalhealth with the power to completely eliminate infectious diseases byactivating the immune system. The ability of the immune system toprotect from disease extends to cancer, as evidenced early on withbacterial mixtures in treating certain cancers to more recently thetherapeutic success of negative co-stimulatory molecule blockade. See,e.g., Mansoor W et al. Br. J. Cancer. 2005; 93(10):1085-91. Thus, byactivating immunity through such methods as cancer vaccines, it ispossible to boost the immune system to enable it to combat cancer moreeffectively.

Malignant melanoma is the deadliest form of skin cancer, and itsincidence is rising. See, e.g., Miller A J et al. N. Engl. J. Med. 2006;355:51-65. Despite the recent development of small molecule inhibitorsand negative co-stimulatory molecule blockade, the prognosis for latestage melanoma is poor, and many of the agents have small therapeuticwindows and result in severe side effects. As more information aboutimmune cells is discovered, researchers have realized that the immunesystem plays a crucial role in preventing cancer including melanoma.Experimental studies have shown that activation of both innate andadaptive immunity can prevent tumor development; therefore thedevelopment of prophylactic vaccine against melanoma may providelong-term protection. A major challenge for designing prophylacticcancer vaccines is to define immunogenic and safe cancer antigens thatcan serve as targets for effective vaccines, including tumor-specificantigens on the tumor.

Some progress has been made in the development of vaccines againstcervical cancer, caused by human papilloma virus. See, e.g., Begue P etal. Bull. Acad. Natl. Med. 2007; 191:1805-16. However, vaccinedevelopment of other types of cancers poses more challenges, since mostcancers are believed not to be caused by infectious agents, but rather,defects in cellular proteins. Since these proteins are very similar tothose found in normal cells, it is difficult to develop vaccinestargeting the cancer cells while sparing normal cells. However, sometumor cells display unusual antigens or cell surface receptors that arerare or absent on the surfaces of healthy cells. See, e.g., Sensi M etal. Clin. Cancer Res. 2006; 12:5023-32. Although cancer vaccines withdefined antigens are commonly used, the use of whole tumor cells incancer immunotherapy is a promising approach and can obviate someimportant limitations in vaccine development. See, e.g., Chiang C L, etal. Semin. Immunol. 2010; 22:132-43. Whole tumor cells are a good sourceof tumor associated antigens and can induce simultaneous Cytotoxic Tlymphocytes and T helper cell activation. Whole cell autologous vaccinestypically consist of irradiated tumor cells that are often transfectedwith granulocyte-macrophage colony-stimulating factor (GM-CSF) or theadoptive transfer of pulsed dendritic cells (DC) or transfected T cells.Inflammatory GM-CSF is a cytokine that plays a critical role inimmunoregulation. See, e.g., Shi Y et al. Cell Res. 2006; 16:126-33.GM-CSF can overcome tumor-induced immune suppression and promotes therecruitment and maturation of specialized antigen-presenting cells (APC)such as DC. DC are potent antigen-presenting cells and play a pivotalrole in T cell-mediated immunity. GM-CSF-mediated activation of APCresults in upregulation of MHC class II, co-stimulatory molecules andcytokine production. See, e.g., Caulfield J J et al. Immunology. 1999;98:104-10. It increases antibody responses and cellular immunity afterimmunization. These combined features have made GM-CSF a commonly usedcytokine to boost anti-tumor immunity. See, e.g., Disis M L et al.Blood. 1996; 88:202-10; Dranoff G et al. Proc Natl Acad Sci USA. 1993;90:3539-43; and Dranoff G. Immunol Rev. 2002; 188:147-54. In a murinemelanoma model, a promising cancer cellular vaccine demonstrated thepotency of prophylactic GM-CSF-transduced autologous tumor cell vaccinesin prevention of tumor outgrowth. See Dranoff G et al. Proc Natl AcadSci USA. 1993; 90:3539-43; and Dranoff G. Immunol Rev. 2002; 188:147-54.However, in these studies, cells required a substantial in vitro geneticmanipulation, leading to high cost and significant regulatory concerns.More importantly, cellular transplantation have been hampered by poorcell viability (<10%), which may prevent long-term GM-CSF secretion andtumor antigen exposition and ultimately vaccine efficacy. See, e.g.,Aguado B A et al. Tissue Eng. Part A. 2012; 18:806-15.

Clinical trials testing the efficacy of whole cell autologous vaccinesfor the treatment of many cancers including melanoma have been conductedwith limited success. An advantage of whole cell irradiated tumorvaccines versus standard peptide or protein vaccination is that a widediversity of patient-specific, tumor associated antigens are presentedon tumor MHC, reducing the concern for cancer immunoediting. However,these techniques present several drawbacks. GM-CSF transfected cellsrequire expensive and laborious gene transfection processes whileadoptively transferred cells may fail to engraft. Further, the clinicalresponse has been limited. Many experimental studies have shown thatactivation of both innate and adaptive immunity can prevent tumordevelopment. The invention overcomes these drawbacks, as theprophylactic vaccine against melanoma provides long-term protectionagainst the disease.

Creating an infection-mimicking microenvironment using an implantableporous scaffold-based device by appropriately presenting exogenouscytokines (for example, GM-CSF) and danger signals for an extended time,in concert with cancer antigens, may provide an avenue to preciselycontrol and enhance the number and timing of dendritic-cell traffickingand activation, in situ. See, e.g., Ali O A et al. N. at Mater. 2009;8:151-8; and Ali O A et al. Sci. Transl. Med. 2009; 1:8ra19. In additionto GM-CSF, another key element of infection that mobilizes and activatesdendritic cells includes ‘danger signals’ related specifically to theinfectious agent. Cytosine-guanosine oligonucleotide (CpG-ODN)sequences, which are uniquely expressed in bacterial DNA, are potentdanger signals that stimulate mammalian dendritic-cell activation anddendritic-cell trafficking. See, e.g., Klinman D M. Nat Rev Immunol.2004; 4:249-58. However, initial studies using implantable tumorlysate-loaded scaffold vaccines required invasive surgeries to beadministered to the patient, limiting sequential vaccinations. See,e.g., Ali O A et al. N. at Mater. 2009; 8:151-8; and Ali O A et al. Sci.Transl. Med. 2009; 1:8ra19. Additionally, tumor lysate can beintrinsically suppressive to DC activation and has been associated witha weaker T cell response and tumor protection than vaccinating withwhole tumor cells. See Chiang C L et al. Semin. Immunol. 2010;22:132-43; and Buckwalter M et al. The American Association ofImmunologists. 2007; 178:48.16.

In order to improve the efficacy of whole cell cancer vaccines whilebeing minimally invasive upon delivery, the invention provides a way tomimic infection and thereby recruit and program immune cells at adistant site away from the tolerogenic milieu of the tumor environment.For this purpose, an injectable polymeric delivery system was designedto enable simultaneously tumor cell-seeding and immunomodulatorsdelivery while serving as a physical, antigen-presenting structure towhich the dendritic cells are recruited, homed, and subsequentlyactivated. Additionally, pre-encapsulation of tumor antigen-associatedcells within a hydrogel improves viability by providing biomechanicalprotection, biochemical survival cues, and scaffolding. See Bencherif SA et al. Proc. Natl. Acad. Sci. USA. 2012; 109:19590-5; Bencherif S A etal. Biomacromolecules. 2009; 10:2499-507; Bencherif S A et al.Biomaterials. 2009; 30:5270-8; Bencherif S A et al. Acta Biomater. 2009;5:1872-83; Bencherif S A et al. J. Biomed. Mater. Res. A. 2009;90:142-53; Bencherif S A et al. Biomaterials. 2008; 29:1739-49; SiegwartD J et al. J. Biomed. Mater. Res. A. 2008; 87:345-58; and Kennedy S etal. Adv Healthc Mater. 2013.

The development of injectable cell-loaded sponge-based materials is apromising direction towards a safe, minimally-invasive, andpatient-compliant vaccine. This is a promising effective cancer vaccinein the prophylactic treatment of melanoma, eliminating the need forinvasive surgeries required implantable scaffolds. The injectable cellloaded vaccine presented herein is a major advancement, particularlywhen the cancer vaccination is achieved without the need for multiple,systemic injections and high total biomolecules loading. The Examplesbelow further describe an exemplary cancer vaccine based on thisconcept, containing a polysaccharide-based material (alginate) andbioactive molecules (GM-CSF and CpG-ODN) that have previously proven tobe successful as immunomodulatory factors in terms of activation ofantigen-presenting cells (APCs) and priming of cytotoxic T cells. SeeKratky W et al. Proc. Natl. Acad. Sci. USA. 2011; 108:17414-9. Forexample, a cancer vaccine of the invention, such as RGD-modifiedsponge-like hydrogels encapsulating simultaneously GM-CSF and CpG-ODN,was first prepared by a cryopolymerization process before being seededwith whole irradiated tumor cells, resulting in the design of the firstsyringe-injectable scaffold-based cancer vaccine.

As demonstrated by the data presented herein, a cryogel vaccinecontaining syngeneic tumor cells, GM-CSF, and CpG-ODN could be injectedusing a large gauge needle, enrich for DC and T cells, and inducepotent, and persistent, anti-tumor immunity. The cryogels were designedto be syringe-injectable and deliver CpG (programming factor) and GM-CSF(programming/recruitment factor) locally and allow for the efficientseeding of melanoma cells. In vivo, the vaccine recruited greater thantwo million DC and three million T cells to the vaccine site and in theprophylactic setting was able to protect 80% of the animals upon initialchallenge and 100% in subsequent re-challenges.

Cancer invokes a tolerogenic milieu that prevents immune clearance,thereby requiring an external boost from immunotherapies to override theimmunosuppression. The active immunotherapy system described hereinstimulates the patient's immune system and promotes an antigen-specificantitumor effect using the body's own immune cells. In addition, thecryogel-vaccine creates a durable antitumor response that protects tumorrecurrence. The strategy in the design of this vaccine was based onmanipulating in situ, dendritic cells recruitment, activation, and theirdispersion to the lymph nodes.

Since the immune system responds to the environmental factors itencounters on the basis of discrimination between self and non-self,many kinds of tumor cells that arise as a result of the onset of cancerare more or less tolerated by the patient's own immune system since thetumor cells are essentially the patient's own cells that are growing,dividing and spreading without proper regulatory control. However, sometumor cells display unusual antigens or cell surface receptors that arerare or absent on the surfaces of healthy cells, and which may beresponsible for activating cellular signal transduction pathways thatcause the unregulated growth and division of the tumor cell. See. e.g.,Sensi M et al. Clin. Cancer Res. 2006; 12:5023-32. The unique andflexible vaccine system described herein is a promising cell-basedimmunotherapy approach for the treatment of melanoma cancer usingtumor-cells. In some examples, distinct tumor cell-associated antigensare extracted from patients, multiplied if needed, integrated into thealginate cryogels and administered back into patients to treat differenttypes of cancer.

Advantageously improving expansion and transplantation, the devices ofthe invention improved cell survival while promoting their proliferation(ex- and in-vivo) after subcutaneous injection of bioluminescent B16-F10cells when integrated to the cryogels, in comparison to free cells(bolus). In addition, in order to increase cellular engraftment withinthe polymeric construct, cryogels were functionalized with RGD peptideto promote adhesion and encapsulation of cells through specificRGD-integrin binding. By this approach, the mechanically robusttumor-cell loaded sponge-like vaccines had the ability to support tumorcell attachment before inoculation as well as after delivery tofacilitate immune cell-trafficking within unfilled void spaces of thelarge interconnected pores of vaccines.

In some embodiments, whole cells (containing tumor antigens), takeneither from the patient (autologous) or from a different patient(allogeneic) are used to stimulate the immune system to mount aresponse. Since the tumor cells are irradiated, they are not harmful;rather, they stimulate the host immune system to recognize the tumorcells. The advantages of whole-cell vaccination over other types ofimmunotherapy that target specific antigens is that multiple and unknowntumor antigens do not need to be identified and may be targeted by boththe innate and adaptive immune system. Furthermore, immunity against asingle antigen may be ineffective in tumors with heterogeneous cellpopulations and carries the risk of inducing tumor antigen escapevariants. See, e.g., Thurner B et al. J. Exp. Med. 1999; 190:1669-78.

For the cell-based vaccines described herein, cell surface antigens areamong the targets naturally accessible to surveillance by the immunesystem, particularly by the cellular immune response. Thus, theinjectable cryogel technology is a suitable system for the whole-cellvaccination, as not only does the matrix increase survivability andretention of antigen-displaying tumor cells at the vaccine site but italso enhances cancer antigen exposure to immune cells. In addition,cultivated tumor cells on the cell-adherent gel vaccines have adifferent morphology from those trypsinized and injected directly, whichlikely contributes to the enrichment of cell surface materials andtherefore an improvement in the effectiveness of the sponge-like cryogelcancer vaccines. Several studies have reported that trypsinization ofcells triggers release of glycoproteins and sugars from the cellsurface, thereby leading to a loss of antigenic properties. See, e.g.,Cook G et al. Nature. 1960; 188:1011-2; Gasic G et al. Nature. 1962;196:170; Uhlenbruck G. Nature. 1961; 190:181; David J et al. J. Exp.Med. 1964; 120:1189-200; Weiss L et al. Exp. Cell Res. 1963; 30:331-8;and Osunkoya B et al. Int. J. Cancer. 1969; 4:159-65. In particular,Molinari and Platt described that polyoma virus-transformed cellstreated with trypsin failed to induce a delayed hypersensitivityreaction against tumor-specific antigens in footpad swelling assays. SeeMolinari J et al. Proc. Soc. Exp. Biol. Med. 1975; 148:991-4. Thus,antigenic targets are present on the surface of cancer cells, and theuse of scaffold-based vaccines likely prevents removal of molecules fromthe cell surface and therefore increases the antigenicity. A majordrawback of unfractionated tumor antigens is the possibility of inducingan autoimmune reactivity to epitopes that are shared by normal tissues.See, e.g., Ludewig B et al. J. Exp. Med. 2000; 191:795-804. However, inclinical trials using lysate or whole tumor cells as the source ofantigen, no clinically relevant autoimmune responses were detected. See,e.g., Li J et al. Cancer Immunol. Immunother. 2001; 50:456-62.

Tumors are recognized by the immune system through unique tumorassociated antigens (TAAs). See, e.g., Buonaguro L et al. Clin VaccineImmunol. 2011; 18:23-34. Although many TAA targets have been identifiedand added in the design of new immunotherapeutic strategies, painstakingwork remains to be done to fully characterize the immunogenicity ofthese emerging antigens in the human, identify the most immunogenicepitopes, and test their role as bona fide tumor rejection antigens thatcan cause tumor regression. A promising alternative to individual TAAsis vaccination using whole tumor cells without defining the antigens.Tumor cells express a whole array of TAAs that are both characterizedand uncharacterized, and this rich source of antigens contains epitopesof both CD8+ cytotoxic T cells (CTLs) and CD4+ T helper cells. Theparallel presentation of both MHC Class I and II restricted antigens canhelp to generate a stronger overall anti-tumor response and long termCD8+ T cell memory via CD4+ T cell help. In addition, it would greatlydiminish the chance of tumor escape compared to using single epitopevaccines. Furthermore, the use of whole tumor cells eliminates the needto define, test, and select for immunodominant epitopes. The tumor cellscan be autologous, i.e., obtained from the patients, or allogeneic“off-the-shelf”. Allogeneic tumor cell lines that share one or evenseveral of the TAAs as autologous tumor cells provide a simpler methodof delivering antigens in tumor immunotherapy. Allogeneic cell lines canbe propagated in large quantities in cell factories and the quality canbe easily assessed and monitored in good manufacturing practicefacilities.

Through the use of distinct tumor cell-associated antigens, the cellularcryogel-based vaccine platform provided by the invention can be adaptedto a variety of cancers. In some cases, active specific immunotherapyinvolves the priming of the immune system in order to generate a T-cellresponse against tumor-associated antigens. One example of the activespecific approach is adoptive T-cell therapy, which involves the ex vivocultivation of T cells with demonstrated activity against a specifictarget cancer antigen. The goal is to increase the frequency of these Tcells to achieve therapeutic levels and then infuse them back into thepatient via injectable alginate-based cryogels. In some embodiments, Tcells are extracted from patients, multiplied in large quantities,integrated into the alginate cryogels and administered back intopatients to treat different types of cancer. This type of T-cell therapywas recently shown to be able to boost the body's ability to fightcancers such as leukemia, lymphoma, melanoma, and breast cancer.However, prior to this invention, T cells were limited by difficultiesin generating enough cells in vitro, T cells usually undergo necroticdeath after transplantation, and a cancer can return if an immuneresponse is not sustained. The vaccine devices described herein aresurprisingly advantageous, e.g., in improving cell expansion andtransplantation, as the devices improve survival of cells whilepromoting their proliferation (ex- and in-vivo) after subcutaneousinjection of bioluminescent B16-F10 cells when integrated to thecryogel, in comparison to free cells (bolus). See, e.g., Bencherif S etal. Proc. Natl. Acad. Sci. USA. 2012; 109:19590-5. This unique andflexible system is a promising cell-based immunotherapy approach for thetreatment of cancers (e.g., breast cancer or melanoma) using allogeneictumor-specific T cells (adoptive T cell therapy).

Cancer Vaccines and Breast Cancer

Breast cancer is a very common disease, affecting approximately one innine women in the western world at some time in their lives. In recentyears, passive immunotherapy has become an effective adjunct for thetreatment of HER2/neu-overexpressing breast cancers. Sometimes, thesecancer cells respond well to agents, such as trastuzumab (monoclonalanti-HER-2/neu protein antibody drug). However, this therapy is onlyeffective in a subset of breast cancers, and patients with late-stagedisease who are often immune-suppressed are unlikely to respond.Furthermore, tumors can evolve to evade the immune response. Therefore,there is a need for a more globally effective, prophylactic vaccine. Tothis end, the invention provides an injectable polymer-derivedprophylactic HER-2/neu-based breast cancer vaccine.

A strategy in the design of this vaccine was based on manipulating insitu dendritic cell recruitment, activation, and their dispersion to thelymph nodes. Cytosine-guanosine oligonucleotide (CpG-ODN) is used as anadjuvant to help stimulate enhanced responses to the vaccine. Thisactive immunotherapy system stimulates the patient's immune system topromote a HER-2/neu-specific antitumor effect using the body's ownimmune cells. In addition, the cryogel-vaccine creates a durableantitumor response that, in some cases, protects against tumorrecurrence.

To create this cryogel vaccine, both components (adjuvant and cytokine)are incorporated into the cryogel matrix (FIG. 5A). These biomoleculesare released in a sustained fashion to recruit and host DCs, andsubsequently present cancer antigens from attenuated cells byirradiation as well as danger signals to activate resident naïve DCs andpromote their homing to the lymph nodes, which is necessary for a robustanti-cancer immune response. As described in the Examples, this cryogelvaccine system is a promising cell-based immunotherapy approach for thetreatment of breast cancer using allogeneic tumor cells. The datapresented herein demonstrates that the HER-2/neu-based cryogel vaccineprovides potent prophylactic protection against mammary cancer.

Injectable Gelatin Cryogels

The performance of biomaterials-based therapies can be hindered bycomplications associated with surgical implant, motivating thedevelopment of materials systems that allow minimally invasiveintroduction into the host. Implantable biomaterials have been proposedto locally deliver or recruit cells, or provide sustained release oftherapeutic molecules for applications such as tissue engineering, drugdelivery, gene therapy, and vaccines. The clinical implantation ofprefabricated biomaterials for these purposes typically requires trainedphysicians, causes patient distress, creates potential scarring, poses arisk of infection, and often causes inflammation at the surgical sitethat may inhibit the performance of the implant. Thus, there is a needfor biomaterials that can be introduced in a minimally invasive manner.Such biomaterials would be of great use in many therapeuticapplications.

Injectable hydrogels have been used as biomaterial implants without theneed for surgery. Many of these materials systems involve the injectionof a polymer solution and subsequent crosslinking of the polymer chainsby chemical or physical means to form a solid. However, the use ofliquid precursors may result in leakage from the implant site tounwanted tissues and poses difficulties in generating the desiredimplant geometry. Although injectable hydrogels have been used asbiomaterial implants without the need for surgery, many of theseinjectable hydrogel have certain disadvantages, such leakage from theimplant site to unwanted tissues and difficulties in generating thedesired implant geometry. Other hydrogels scaffolds are not easilyremodeled and/or degraded by cells locally—as such, their ability tointegrate with host tissue is limited. Some of these hydrogel scaffoldsalso require modification of the alginate polymer with cell adhesivepeptides in order to allow cell attachment, which requires additionalsynthesis steps. Thus, the hydrogels of the invention have distinctadvantages over previously described injectable hydrogels. Thisinvention provides a preformed hydrogel scaffold with a defined geometryand microstructure that can be introduced to the body in a minimallyinvasive manner through a conventional needle. See, e.g., WO2012/149358, incorporated herein by reference. These hydrogels areformed by cryopolymerization of a material such as methacrylatedalginate using radical polymerization at sub-zero temperatures. Thesescaffolds are capable of delivering cells and biomolecules in anon-invasive manner. To allow for cell attachment, these alginatehydrogels are modified with cell adhesive peptides.

The cell-adhesive and degradable gelatin cryogel scaffold that can beinjected through a conventional needle while maintaining a predefinedgeometry and architecture. These gelatin cryogels have certainadvantages over other hydrogels or cryogels (such as alginate cryogels).Gelatin is a heterogenous mixture of polypeptides that is derived fromcollagen by partial hydrolysis. Collagen is an insoluble fibrous proteinthat occurs in vertebrates and is the main component of connectivetissues and bones. For example, the collagen used to make gelatin isisolated from the connective tissues and bones of animals, e.g., fromskin and bones. Gelatin is commercially available at a pharmaceuticalgrade. Exemplary types of gelatin include gelatin derived from porcineskin, beef skin, or bone. For example, gelatin is derived by acidtreatment of collagenous material (also called Type A gelatin) or alkalitreatment of collagenous material (also called Type B gelatin). Otherexamples of gelatin include recombinant human gelatin (e.g., availablefrom Fibrogen, Inc., www.fibrogen.com/recombinantgelatin) and lowendotoxin gelatin preparation from animal origin is another example of agelatin (e.g., available from Nitta Gelatin NA Inc.,http://nitta-gelatin.com/bematrix-low-endotoxin-gelatin/). In somecases, the cryogels of the invention contain Type A gelatin, such asfrom porcine skin.

The gelatin used in the cryogels has a molecular weight range of 20,000to 250,000 g/mol (e.g., 20,000 to 200,000 g/mol, 50,000 to 150,000g/mol, or 75,000 to 100,000 g/mol). The amino acid composition ofgelatin contains about 0.1 to about 0.5% (e.g., about 0.2%) tyrosine andabout 10% to about 50% (e.g., about 30%) glycine. For example, gelatincontains 20-35% glycine (e.g., 24-32% glycine), 10-20% proline (e.g.,14-18% proline), 10-20% (e.g., 12-16%) hydroxyproline, 8-15% (e.g.,10-12%) glutamic acid, 5-15% (e.g., 8-12%) and alanine. For example,gelatin is water-soluble. In some embodiments, an elemental makeup ofgelatin includes 45-55% (e.g., 50%) carbon, 5-10% (e.g., 7%) hydrogen,15-20% (e.g., 17%) nitrogen, and 20-30% (e.g., 25%) oxygen.

Gelatin is a material with inherent cell-responsive (e.g., cell bindingand enzymatically degradable) elements that can improve the performanceof cryogel implants by allowing direct cell attachment and localremodeling. Derived from collagen, gelatin contains inherent peptidesequences that facilitate cell adhesion and enzymatic degradation.Additionally, its low cost, lack of immunogenicity, and safety record inmedicine (e.g., as a hemostatic agent or blood volume expander) makesgelatin an attractive implantable biomaterial. Modification of gelatinwith pendant methacrylate groups (GelMA) allows crosslinked hydrogels tobe formed using radical polymerization, which have been used extensivelyin cell culture and tissue engineering studies. In some cases, thegelatin (e.g., Type A gelatin) is methacrylated, i.e., pendantmethacrylate groups are added primarily to the free amines of gelatin byreaction with methacrylic anhydride. For example, the methylacrylatedgelatin contains a degree of substitution of at least 50%, at least 60%,at least 70%, at least 80%, at least 90% (e.g., about 79%).

The fabrication and characterization of scaffolds formed bycryopolymerization of GelMA (cryoGelMA) are described herein. Inparticular, the bulk mechanical behavior, structure, and degradation ofthe cryoGelMA gels, as well as the ability of these scaffolds tofacilitate cell attachment, proliferation, and survival werecharacterized. Additionally, the ability of gelatin cryogels to locallydeliver a chemoattractant protein, recruit host cells, and undergocell-mediated degradation in vivo is also reported herein.

The results presented herein show that porous cryoGelMA gels of adefined shape can be injected through a conventional needle and regaintheir geometry and architecture after ejection from the needle bore.This is a result of the thin-walled and highly porous structure of thesegels imparted through cryopolymerization. Application of a mechanicalstress during injection causes rapid efflux of water from theinterconnected pores and collapse of the scaffold structure. Removal ofstress after injection releases stored elastic energy and causes waterinflux into the hydrophilic gel, resulting in recovery of the cryogelshape. Previously reported injectable gelatin-based gels have relied onliquid prepolymers that require crosslinking agents to allow in situgelation. See, e.g., Kuwahara K. et al. Tissue Eng Part C Methods 2010;16:609-18; and Sakai S. et al. Biomaterials 2009; 30:3371-7. The use ofa liquid precursor generally does not allow a defined geometry ormicroarchitecture to be created in the resulting gel, and prepolymerleakage to unwanted tissues may occur prior to gel formation. CryoGelMAgels of predefined shape and porosity can be implanted in a highlylocalized manner at any site that can be safely accessed with a needle.

Also, cryoGelMA gels are cell and tissue compatible. CryoGelMA gelsallowed cell attachment, spreading, proliferation, and sustainedviability of fibroblasts in vitro. In vivo implanted gels produced mildacute inflammation followed by a foreign body response at the scaffoldperiphery, which is typical of many biomaterials. See, e.g., Mikos A. etal. Adv Drug Deliv Rev 1998; 33:111-39. Nanoporous GelMA hydrogels arecompatible in cell culture applications for a variety of mouse and humancell types in 2D and 3D. See, e.g., Nichol J. et al. Biomaterials 2010;31:5536-44; Benton J. et al. Tissue Eng Part A 2009; 15:3221-30; Qi H.et al. Adv Mater 2010; 22:5276-81; Ramón-Azcón J et al. Lab Chip 2012;12:2959-69; and Aubin H et al. Biomaterials 2010; 31:6941-51. Inaddition, cell-laden nanoporous GelMA hydrogels introduced into athymicnude mice elicit minimal inflammation and cell infiltration. See, e.g.,Nichol J et al. Biomaterials 2010; 31:5536-44; and Lin R-Z et al.Biomaterials 2013; 34:6785-96. The cell and tissue compatible nature ofporous cryoGelMA gels are favorable for applications where celltrafficking within the scaffold is important, such as cell delivery andrecruitment.

The results of both in vitro and in vivo degradation studies describedherein show that cryoGelMA gels are enzymatically degradable.Collagenase preparations, mammalian MMP-2 and -9 were capable ofdegrading cryoGelMA gels in vitro, indicating that the enzymaticdegradability of gelatin was preserved after modification withmethacrylate pendant groups and cryopolymerization. Controlled releaseof GM-CSF from the scaffold resulted in complete scaffold infiltrationby immune cells, increased MMP activity, and accelerated scaffolddegradation. These results are consistent with the role of GM-CSF as amediator of immune cell recruitment, and as an inducer of MMPproduction. CryoGelMA surprisingly degraded at a slower rate in thepresence of collagenase type II in vitro than has been previouslyreported for nanoporous GelMA hydrogels. See, e.g., Hutson C et al.Tissue Eng Part A 2011; 17:1713-23. Also surprisingly, cryoGelMAdegradation in vivo was also substantially slower than reported forother implantable gelatin formulations. See, e.g., Chen Y-C et al. AdvFunct Mater 2012; 22:2027-39; Kang H-W et al. J Bioact Compat Pol 1999;14:331-43; and Tabata Y et al. J Control Release 1994; 31:189-99. Thisenhanced stability of cryoGelMA may be due to an overall higher degreeof crosslinking and entangled polymer network density achieved withcryopolymerization relative to conventional bulk hydrogel formation.CryoGelMA gels can undergo cell-mediated remodeling at the injectionsite, alleviating the need for surgical explant in certain applications.

CryoGelMA gels also provide localized controlled release of proteins.For example, cryoGelMA releases GM-CSF in a sustained manner, andsubcutaneously injected GM-CSF-cryoGelMA releases bioactive GM-CSF,which causes immune cell recruitment to the scaffold. CryoGelMA allowsthe encapsulation of proteins in a single step that occurs concurrentlywith gel formation. This is an advantage over commonly usedprotein-loaded gelatin hydrogel fabrication schemes that usecrosslinking agents that cause protein deactivation during directencapsulation (e.g., glutaraldehyde). See, e.g., Young S et al. JControl Release 2005; 109:256-74. Using gelatin as the scaffold materialallows release of molecules of various net charges based on theprocessing used for gelatin fabrication and/or through chemicalmodifications to alter the electrostatic properties of gelatin.Additionally, the tightly packed, entangled, and highly crosslinkedpolymer network formed by phase separation during cryopolymerizationprovides an increased physical barrier to protein release relative toconventionally formed hydrogels. Enzymatic degradation of the cryogelwalls by recruited host cells facilitates release of entrapped proteins.These properties allow cryoGelMA to provide cell-triggered release oftherapeutic proteins at sites of disease.

The gelatin cryogels described herein (e.g., the cryoGelMA) are suitablefor use as a cryogel vaccine (e.g., to vaccinate against a cancer orinfectious disease) as described in detail herein. For example, thegelatin cryogels are loaded with GM-CSF and CpG-ODN as well as anantigen (e.g., a cancer cell antigen, such as an irradiated orattenuated cancer cell).

The following experimental methods were used to characterize the cryogelvaccines for melanoma described herein.

Materials

UP LVG sodium alginate with high gluronate content was purchased fromProNova Biomedical; 2-morpholinoethanesulfonic acid (MES), sodiumchloride (NaCl), calcium chloride (CaCl2) sodium hydroxide (NaOH),N-hydroxysuccinimide (NHS),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC),2-aminoethyl methacrylate hydrochloride (AEMA), and acetone werepurchased from Sigma-Aldrich. ACRL-PEG-NHS (3.5 kDa) was purchased fromJenKen Technology. Rhodamine-labeled polylysine was obtained from NanocsInc. Alexa Fluor 488-phalloidin and 4′,6-diamidino-2-phenylindole (DAPI)were purchased from Life Technologies. The integrin binding peptide(Gly)4-Arg-Gly-Asp-Ala-Ser-Ser-Lys-Tyr (SEQ ID NO: 1) was custom made byCommonwealth Biotech. Regular B16-F10 cells (ATCC) and GM-CSF transducedB16-F10 cells (kindly provided by the Dranoff's lab at Dana FarberCancer Institute) were cultured in Dulbecco's modified Eagle medium(DMEM) supplemented with 10% (vol/vol) FBS and 1% (vol/vol)penicillin-streptomycin, all obtained from Invitrogen.

Chemical Modification

MA-alginate was prepared by reacting alginate with AEMA. Sodium alginate(1 g) was dissolved in a buffer solution [0.6% (wt/vol), pH ˜6.5] of 100mM Mes. NHS (1.3 g) and EDC (2.8 g) were added to the mixture toactivate the carboxylic acid groups of alginate. After 5 min, AEMA (2.24g; molar ratio of NHS:EDC:AEMA=1:1.3:1.1) was added to the product andthe solution was stirred at RT for 24 h. The mixture was precipitated inacetone, filtered, and dried in a vacuum oven overnight at RT. ¹H NMRwas used to characterize chemical modification of alginate and degree offunctionalization of MA-alginate (FIG. 10).

Cryogel Vaccine Fabrication

Macroporous matrices were synthesized by redox-induced free-radicalpolymerization of MA-alginate in water. ACRL-PEG-G4RGDASSKY (SEQ ID NO:2) was synthesized according to Bencherif S et al. Biomaterials. 2008;29:1739-49, incorporated herein by reference. Alginate cryogel vaccineswere synthesized by mixing 23 mg [2.3% (wt/vol)] of MA-alginatemacromonomer in deionized water with tetramethylethylenediamine (TEMED)[0.5% (wt/vol)] and ammonium persulfate (APS) [0.25% (wt/vol)]. CpG-ODN1826, 5′-TCC ATG ACG TTC CTG ACG TT-3′ (SEQ ID NO: 3), (Invivogen) andGM-CSF (PeproTech) were added to the polymer solution priorcryopolymerization. Fabrication conditions were chosen to allow thesolution to freeze before the gelation takes place. More specifically,the precursor solution was precooled to 4° C. to decrease the rate ofpolymerization before freezing, and once the initiator was added to theprepolymer solution, the solution was quickly poured into a precooled(−20° C.) Teflon mold. After a complete incubation period of 17 h, thegels were heated to RT to remove ice crystals and washed with deionizedwater. Cell-adhesive cryogels were synthesized using ACRL-PEG-G4RGDASSKY(SEQ ID NO: 2) as a comonomer [0.8% (wt/vol)] during the polymerization.Conventional hydrogels were cross-linked for 30 min at RT for ahomogeneous gelation.

Cell Seeding and Incubation of Cellular Cryogel Vaccines

Before seeding cells, the cryogels were treated with 70% ethanol andwashed with PBS. The cryogels were mechanically compressed to partiallyremove pore water under sterile conditions before cell seeding. Briefly,B16-F10 cells were suspended in complete culture medium (DMEMsupplemented with 10% FBS and 1% penicillin-streptomycin). Priorseeding, tumor cells were irradiated by receiving 3500 rads (1 rad=0.01Gy) from a 137Cs source discharging 208 rads/min for 17 min. Twentymicroliters of a cell suspension (10⁷ cells/mL) were added in a dropwisemanner on top of each square-shaped cryogel and the cell-loaded cryogelswere cultured in FBS-supplemented media for 6 h (37° C. in 5% CO₂environment). Cell distribution was noted to be homogeneous throughoutthe gel construct.

Controlled Release of Immunomodulator Factors

To determine the incorporation efficiency and release kinetics ofCpG-ODN and GM-CSF from cryogel vaccines, standard release studies werecarried out. GM-CSF and CpG-ODN releases in the supernatant weredetected by ELISA (Invitrogen) and OliGreen assay (Invitrogen),respectively.

Hydrogel Characterization

Structural analysis of the macroporous gel-vaccine was performed using aLEO 982 scanning electron microscope (SEM) (LEO Electron Microscopy). Toprepare the samples, cryogels in the frozen state following cryogelationwere lyophilized and sectioned for observation. The average size ofpores in cryogels was calculated by averaging the diameters of the poresin the gels observed by SEM. The distribution of cells within thescaffolds was visualized with an inverted laser scanning confocalmicroscope (Leica SP5 XMP, Germany). High-resolution image stacks werecollected with 300-nm separation between slices (z-stacks) for the 3Dreconstruction of the entire scaffold and visualization of cell-matrixinteractions.

Generation of Bone Marrow-Derived DC (BMDC) and In Vitro DC ActivationAssays

BMDC from bone marrow progenitors were generated by the followingprotocol. Murine bones were placed in 70% ethanol for 2 min andsubsequently washed in PBS. Both bone ends were cut off, and the marrowwas flushed out with RPMI 1640 medium (Gibco, Grand Island, N.Y.). Thered cells were lysed with ammonium chloride (0.45 M). The cells werecentrifuged for 10 min at 1,500 rpm and 2×10⁵/ml cells were cultured for10 d in Petri dishes in 10 ml of complete medium (RPMI 1640 mediumsupplemented with 10% FBS, 1% penicillin-streptomycin, 2 mM L-glutamine,5×10⁻⁵ M 2-mercaptoethanol, and 15%-30% of mouse GM-CSF). To evaluateviability and activation induction of BMDC from CpG-ODN loaded cryogelvaccines, two square-shaped blank scaffolds or scaffolds containingCpG-ODN (5′-TCC ATG AGC TTC CTG AGC TT-3′) (SEQ ID NO: 4) were incubatedwith bone morrow DC in complete culture medium for 1 d (RPMI 1640 mediumsupplemented with 10% FBS and 1% penicillin-streptomycin). A live/deadassay was performed to evaluate BMDC viability in the presence ofcryogels. Briefly, isolated BMDC in triplicate were incubated with thelive/dead assay dye solution (Molecular Probes) containing 0.5 μL ofcalcein-AM and 2 μL of ethidium homodimer-1 in 1 mL of PBS. After 30 minincubation, the cells were rinsed with PBS, and cell viability wasquantified by flow cytometry (FACS). Yield evaluation and induced BMDCmaturation was quantified and tested by FACS, respectively. Thefollowing antibodies conjugated to fluorescent markers (Proimmune) wereused for surface stainings: MHC II (2 G9/PE), CD86 (B7-2, GL1, ratIgG2a), CD11c (N418, hamster IgG). For purified antibodies, theappropriate anti-TNP isotype controls were used. The IL-12 concentrationin the cell-culture supernatant was then analyzed with ELISA (R&Dsystems), according to the manufacturer's instructions.

Minimally Invasive Delivery of Cryogel Cancer Vaccines and CellRecruitment

Female C57BL/6J mice (n=10; The Jackson Laboratory), 4-6 wk of age, wereanesthetized with 2.5% isoflurane using an inhalation anesthesia system(E-Z Anesthesia; Euthanex). Each mouse received a 2 s.c. dorsalinjections of cryogel vaccines suspended in 0.2 mL of PBS by means of a16-gauge needle under the dorsal panniculus carnosus. To visualize thecell-recruitment and homing capacity of cryogels, mice were sacrificed,and the explanted scaffolds were embedded in paraffin and 5 μm sectionswere stained with hematoxylin and eosin (H&E) for histological analysis.To quantify dendritic cell recruitment, explanted cryogels were digestedin collagenase type II (250 U/ml; Worthington) that was agitated at 37°C. for 45 min. The cell suspensions were then poured through a 40 mmcell strainer to isolate cells from scaffold particles, and the cellswere pelleted and washed with cold PBS and counted with a Z2 coultercounter (Beckman Coulter). To assess DC infiltration, cells isolatedfrom alginate sponges were then stained with primary antibodies(Proimmune) conjugated to fluorescent markers to allow for analysis byflow cytometry. Allophycocyanin (APC)-conjugated CD11c andAPC-conjugated CD11c stains were conducted for DC infiltration analysis.Animal work was performed under a protocol approved by the HarvardStanding Committee on Animals in compliance with the National Institutesof Health guidelines.

In Situ Identification of DC Subsets and T Cells

Blank alginate sponges and sponges containing 1.5 μg of GM-CSF incombination with 100 μg of CpG-ODN were injected into subcutaneouspockets on the back of 6- to 9-week-old male C57BL/6J mice. To analyzeDC recruitment, scaffolds were excised at various time points anddigested the ingrown tissue into single-cell suspensions with acollagenase type II solution (250 U/ml; Worthington) that was agitatedat 37° C. for 45 min. The cell suspensions were then poured through a 40mm cell strainer to isolate cells from scaffold particles, and the cellswere pelleted and washed with cold PBS and counted with a Z2 coultercounter (Beckman Coulter). To assess DC infiltration and activation,subsets of the total cell population isolated from alginate sponges werestained with primary antibodies (Proimmune) conjugated to fluorescentmarkers to allow for analysis by flow cytometry.

Allophycocyanin (APC)-conjugated CD11c (DC marker) and phycoerythrin(PE)-conjugated CD86 (B7, costimulatory molecule) stains were conductedfor DC recruitment analysis, and APC-conjugated CD11c, and PE-conjugatedMHCII stains were conducted for DC programming analysis. To furtherdelineate the presence of specific DC subsets, cells were stained withAPC-conjugated CD11c and PE-conjugated PDCA-1 (pDC marker),APC-conjugated CD11c and PE-conjugated CD8 (CD8 DC), or APC-conjugatedCD11c and FITC-conjugated CD11b (CD11b DC). To assess T cellinfiltration, PE-Cy7-conjugated CD3 stains were performed in conjunctionwith APC-conjugated CD8a (CD8 T cells), FITC-conjugated CD4 (CD4 Tcells), and PE-conjugated FoxP3 (Treg) and analyzed with flow cytometry.Cells were gated according to positive FITC, APC, and PE with isotypecontrols, and the percentage of cells staining positive for each surfaceantigen was recorded. To determine the cytokines (IL-12, IFN-γ, etc)concentration at the cryogel vaccine site, adjacent tissue was excisedand digested with tissue protein extraction reagent (Pierce). Severalcytokines concentrations in the tissue were then analyzed with aBio-Plex Pro™ Mouse Cytokines 23-plex Assay, according to themanufacturer's instructions.

Prophylactic Immunization, Long-Term Protection, and Toxicology

Two square-shaped RGD-containing alginate sponges pre-cultured withirradiated melanoma tumor cells and each loaded with GM-CSF (1.5ug/scaffold) and CpG-ODN (50 ug/scaffold) were injected subcutaneouslyinto each sides of the lower flank of C57BL/6J mice (n=10/group). Forcontrol groups, mice were also injected with blank cell-seeded alginatesponges as well as sponges loaded with one single immunomodulatorfactor, either GM-CSF or CpG-ODN. Additionally, cryogel cancer vaccineswere compared with a common cell-based vaccine. Therefore, a group ofmice were vaccinated with 5×10⁵ irradiated (3500 rads) GM-CSF-transducedB16-F10 cells, as described previously. See Dranoff G et al. Proc. Natl.Acad. Sci. USA. 1993; 90:3539-43; and Dranoff G. Immunol.

Rev. 2002; 188:147-54, incorporated herein by reference. Animals werechallenged 6 days later with a subcutaneous injection of 1×10⁵ B16-F10melanoma cells in the back of the neck. To assess long-termimmunological response to melanoma vaccines, surviving mice in eachgroup (n>3) were tumor rechallenged following 126 days post first tumorchallenge with 1×10⁵ B16-F10 melanoma cells. Animals were monitored forthe onset of tumor growth (approximately 1 mm³) and sacrificed whenchallenge tumors reached 20 mm (longest diameter) or severe ulcerationor bleeding developed. Following 18 months after initial tumorchallenge, several surviving immunized mice from successive tumorinoculations (day 6 and day 96 post vaccination) were euthanized.Tissues from the major organs (heart, liver, spleen, lungs, kidney,brain, lymph nodes, pancreas, small intestine, colon, stomach) andexplanted implants were fixed in a 10% neutral buffered formalinsolution, resuspended in 70% ethanol and sent to Mass Histology Service(Worcester, Mass.) for toxicology study.

Statistical Analysis

All values in the present study were expressed as mean±1 SD unlessotherwise noted. The significant differences between the groups wereanalyzed by a Student's t-test, ANOVA, and log rank test. Differenceswere considered significant at P<0.05.

The following experimental methods were used to characterize the gelatincryogels described herein.

Mice

C57BL/6J and C57BL/6J-Tyr^(c-2J) mice (female, aged 6-8 weeks; JacksonLaboratories) were used in the experiments described herein.

Methacrylated Gelatin Synthesis

Methacrylated gelatin (GelMA) was synthesized (FIG. 26A) by allowingType A porcine skin gelatin (Sigma) at 10% (w/v) to dissolve in stirredDulbecco's phosphate buffered saline (DPBS; GIBCO) at 50° C. for 1 h.Methacrylic anhydride (Sigma) was added dropwise to a final volume ratioof 1:4 methacrylic anhydride:gelatin solution. This resulted in GelMAwith a degree of substitution of 79% (FIG. 27). The solution was stirredat 50° C. for 1 h, and then diluted 5× with DPBS. The resulting mixturewas dialyzed in 12-14 kDa molecular weight cutoff tubing (Spectrum Labs)for 4 d against distilled water (dH₂O) with frequent water replacement.The dialyzed solution was lyophilized, and the resulting GelMA wasstored at −20° C. until use. Rhodamine-labeled GelMA, created from thereaction of GelMA with NHS-rhodamine (Thermo Scientific), was purifiedusing an identical dialysis and lyophilization process.

Gelatin Cryogel Preparation

Cryogels were formed by dissolving GelMA in dH₂O to the final desiredconcentration in the presence of 0.5% (w/v) ammonium persulfate (APS;Bio-Rad) and 0.1% (w/v) tetramethylethylenediamine (TEMED; Bio-Rad).This prepolymer solution was pipetted into cylindrical (5 mm diameter, 2mm thickness) polystyrene molds and placed in a freezer set to −12° C.(FIG. 26B). Cryopolymerization was allowed to proceed for 18 h, and theresulting cryogels were thawed and hydrated in dH₂O prior to use.

Interconnected Porosity

To test for cryogels for interconnected porosity, scaffolds were firstthawed and hydrated for 1 d. Hydrated scaffolds were weighed on a scale,and a Kimwipe was lightly applied to the scaffold surface for 30 s towick away loosely held water, and the mass was again recorded. Theinterconnected volume was calculated as the mass of water wicked awaydivided by the total hydrated mass.

Scanning Electron Microscopy

For scanning electron microscopy, cryogels were serially transitionedfrom dH₂O into absolute ethanol with 20 min incubations in 30, 50, 70,90, and 100% ethanol solutions. Samples were incubated inhexamethyldisilazane (Electron Microscopy Sciences) for 10 min and driedin a desiccator for 1 h. Dried cryogels were adhered onto sample stubsusing carbon tape and coated with a platinum/palladium in a sputtercoater. Samples were imaged using secondary electron detection on a CarlZeiss Supra 55 VP field emission scanning electron microscope (SEM).Cell-laden cryogels were fixed in 4% paraformaldehyde (PFA) and preparedfor SEM as described above. Images were false-colored in Adobe PhotoshopCS6 to highlight cells.

2-Photon Microscopy

To characterize the hydrated cryogel structure, rhodamine-GelMA cryogelswere placed in dH₂O in a 35 mm glass-bottom culture plate (MatTek), andimaged on a Leica SP5 inverted laser scanning confocal microscope.2-photon excitation was achieved using a Chameleon Vision 2 pulsedinfrared (IR) laser (Coherent) at 820 nm, and fluorescence emission wascollected through a 565-605 nm bandpass filter by a non-descanneddetector. For imaging of cell-laden cryogels, cells were first labeledwith 5-chloromethylfluorescein diacetate (CMFDA) according to themanufacturer's instructions (Molecular Probes) prior to seeding onscaffolds. After cell attachment, cells were fixed with 4% PFA in DPBS,and cell nuclei were stained with Hoescht 3342 (Molecular Probes). For3-color imaging of cell-laden rhodamine-cryoGelMA scaffolds, the IRlaser was tuned to 800 nm and Hoescht 3342, CMFDA, and rhodamine-GelMAwere detected through 430-480 nm, 500-550 nm, and 565-605 nm bandpassfilters, respectively.

Bulk Cryogel Imaging

Bright field and fluorescence images of bulk cryogels were acquiredusing a Zeiss Axio Zoom V16 stereomicroscope. To capture high-speedcryogel injection videos, a Hamamatsu Orca-Flash 4.0 sCMOS camera wasused at 200 frames per second.

Cells and Cryogel Seeding

NIH 3T3 cells were cultured in Dulbecco's Modified Eagles Medium (DMEM)with 10% (v/v) fetal calf serum, 100 U/ml penicillin, and 100 μg/mlstreptomycin (Gibco) at 37° C. in a 5% CO2 atmosphere. Prior to seedinginto cryogels, cells were harvested using a non-enzymatic celldissociation solution (Sigma) and resuspended at 10⁷ cells/ml incomplete medium. Cryogels were dehydrated by wicking with a Kimwipe, andrehydrated in 100 μl of cell suspension in a 2 ml round-bottomcentrifuge tube. Tubes were incubated at 37° C. for 45 min to allow forcell attachment. Gels were placed directly in complete medium followingincubation, and cultured on an orbital shaker at 80 rpm.

In Vitro Cell Viability

To assess viability, cells were retrieved after culture on cryogelsusing 0.05% trypsin/EDTA at 37° C. for 45 min. Scaffolds weremechanically disrupted with a pipette, and filtered through a 70 μm cellstrainer. Cells were stained using the Muse Count and Viability AssayKit and analyzed using a Muse Cell Analyzer (EMD Millipore). To assessmetabolic activity, cryogels were transferred to new wells with 10%AlamarBlue (AbD Serotec) in cell culture medium and incubated for 6 h.The reduction of the substrate was assessed as described in themanufacturer's instructions.

Cryosection Preparation and Staining

Prior to freezing and sectioning, samples were fixed in 4% PFA in DPBSat room temperature for 20 min. The gels were washed three times in DPBSand placed in a 30% (w/v) sucrose in DPBS solution overnight at 4° C.Cryogels were then incubated at room temperature in a 15% (w/v)sucrose+50% OCT compound (Tissue-Tek) for 4 h, and then neat OCT for 4h. Gels were frozen in OCT on dry ice. The frozen blocks were sectionedat −20° C. into 20 am sections on a Leica CM1950 cryostat. F-actin wasstained using Alexa Fluor 488 phalloidin (Molecular Probes). Sampleswere mounted in Prolong Gold Antifade Reagent with DAPI (MolecularProbes), coverslipped, and imaged on a Zeiss LSM 710 upright confocalmicroscope. To visualize proliferation within the cryogels, cells werepulsed with 5-ethynyl-2′-deoxyuridine (EdU; Molecular Probes) for 6 h,fixed, cryosectioned, and stained according to the manufacturer'sinstructions.

Gelatin Zymography

Zymography was performed using a 10% polyacrylamide gel, containing 0.1%(w/v) gelatin or GelMA using standard protocols. Recombinant human(Chemicon) and murine (R&D Systems) pro-MMP-2 and -9 were used to assessGelMA zymogram degradability. For zymography of in vivo implant sites,scaffolds were mechanically disrupted using a 20 G needle in 300 μl oflysis buffer composed of 0.025 M Tris-HCl, pH 7.5, 0.1 M NaCl, 1% v/vIGEPAL CA-630, 10 μg/ml aprotinin, 2 μg/ml leupeptin, and 4 mMbenzamidine (Sigma). Samples were vortexed briefly, incubated for 30 minon ice, and centrifuged at 16 000×g to remove debris. The proteincontent of the resulting lysate was quantified using a bicinchoninicacid assay (Thermo Scientific), and samples were diluted to an equalprotein concentration prior to loading for zymography.

In Vitro Cryogel Degradation

Cryogels created using rhodamine-GelMA were incubated with 25 U/mlcollagenase type II (Worthington) in DPBS and were placed on an orbitalshaker at 80 rpm and 37° C. The collagenase solution was collectedperiodically and completely replaced with fresh enzyme solution.Fluorescence of the supernatants with excitation at 550 nm and emissionat 580 nm were compared to a standard curve of rhodamine-GelMA alsoprepared in collagenase. To test the degradability of cryogels bymammalian enzymes, rhodamine-cryoGelMA gels were treated withrecombinant murine and human MMP-2 and -9. Recombinant proenzymes wereactivated with 2.5 mM p-aminophenylmercuric acetate (Calbiochem) inzymogram development buffer for 3 h at 37° C., and each gel wasincubated in 100 μl of buffer containing 1 μg of enzyme for a subsequent18 h. Fluorescence signal from the supernatants was measured on a platereader, and normalized to fluorescence signal from gels incubated inzymogram development buffer only.

In Vivo Imaging

In vivo imaging studies were performed using an IVIS Spectrum system(PerkinElmer). Living Image 4.0 (PerkinElmer) software was used forquantitative analysis of all images. Cryogels were injectedsubcutaneously into mice using a 1 ml syringe equipped with a 16 Gneedle in 200 μl DPBS. To monitor gel degradation in vivo,rhodamine-GelMA was injected subcutaneously in the flank ofC57BL/6J-Tyr^(c-2J) mice and fluorescence signal was monitoredlongitudinally with excitation and emission measured at 570 nm and 620nm, respectively. The opposite flank was injected with an unlabeledcryogel, and the fluorescence signal from this site was subtracted tocorrect for baseline.

To visualize local MMP activity at the cryogel site, mice weremaintained for two weeks prior to imaging on an alfalfa-free purifiedrodent diet (Harlan) in order to reduce food autofluorescence. Next,mice were injected with rhodamine-labeled cryogels, and injectedintravenously with MMPSense 750 FAST 1 w later. 6 h post-injection,fluorescence signal was measured at 745 nm excitation and 800 nmemission. Baseline fluorescence signal at the scaffold site prior toMMPSense 750 FAST injection was subtracted for quantitative analysis.

Histology of Injected Cryogels

Cryogels injected in C57BL6J mice were retrieved along with thesurrounding 1×1 cm skin samples and fixed overnight in 10% neutralbuffered formalin. Samples were embedded in paraffin and sectioned at 7am thickness by the Harvard Rodent Histopathology Core. Hematoxylin andeosin (H&E) staining was performed to assess inflammation and cellrecruitment at the implant site.

GM-CSF Release

5 μg of recombinant murine granulocyte macrophage colony-stimulatingfactor (GM-CSF) (Peprotech) was incorporated per cryogel by directencapsulation prior to polymerization. Gels were retrieved while frozenand placed in 1% BSA fraction V (Roche) in DPBS and placed on an orbitalshaker at 80 rpm. The complete supernatant was recovered periodicallyand frozen until it was analyzed using a murine GM-CSF ELISA (R&DSystems). The amount of GM-CSF in the supernatant of the thawingsolution after 1 h was considered unencapsulated and was used tocalculate the encapsulation efficiency.

Quantification of In Vivo Cell Recruitment

Blank and GM-CSF-releasing gelatin cryogels were thawed and hydrated for1 h prior to injection. After the desired implantation period, scaffoldswere retrieved and digested in 250 U/ml type II collagenase in DPBS for45 min at 37° C., and counted using a Muse Cell Analyzer.

¹H-NMR

GelMA and control dialyzed gelatin were dissolved in deuterium oxide(D₂O; Sigma) at 15% (w/v) at 80° C. ¹H-NMR spectra with samplesequilibrated at 60° C. on a 600 MHz NMR spectrometer (Varian). Data wereFourier transformed, baseline corrected, and referenced to D₂O. Tocalculate the degree of substitution, the integral of the peakscorresponding to the methylene protons of the methacrylate groups werecompared to the integral of the aromatic side chain protons, aspreviously described. See, e.g., Nichol J W. Biomaterials 2010;31:5536-44.

Mechanical Testing

Cryogel samples were hydrated for one day prior to mechanical testing.Samples were compressed at a rate of 1 mm/min on an Instron 3342mechanical tester equipped with a 50 N load cell. The Young's moduluswas calculated from the linear region of the resulting stress-straincurve between 0-5% strain. Fracture was identified as an abruptdisruption in the rate of increase of stress with strain.

Live Dead Imaging

Cells on cryogels were stained with a Live/Dead kit (Molecular Probes)to assess viability. Media was removed from the culture wells, and gelswere washed with DPBS. DPBS containing 2 am calcein AM and 4 am ethidiumhomodimer-1 was added to the wells and a 20 min incubation was performedat 37° C. Scaffolds were imaged on a Zeiss Axio Zoom V16stereomicroscope.

BrDU Incorporation Assay

Cryogel cell cultures were pulsed with 30 μM bromodeoxyuridine (BrDU)for 4 h. Cells were retrieved using trypsin, fixed and permeabilizedwith Cytofix/Cytoperm (BD Biosciences), additionally permeabilized with0.5% Triton X-100, treated with DNAse (1 μg/10⁶ cells), stained withanti-BrDU APC antibody (BU20A; eBioscience), and analyzed by flowcytometry using a BD LSRFortessa cell analyzer.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Example 1: Injectable Biodegradable Preformed Macroscopic Geometric Gels

The compositions and methods described herein provide hydrogels forminimally invasive delivery of shape memory scaffolds for in vivoapplications. This method has demonstrated highly efficient andreproducible fabrication of injectable shape-defined macroporousscaffolds. Although only one type of covalently alginate-basedcrosslinked gel system was evaluated herein, the material performance isreadily manipulated by altering its composition, formulation, anddegradation profile. The formation of specific shapes and structuralstability are desirable characteristics for shape-defined materials, andthe most important requirement of these types of materials for minimallyinvasive therapies is the ability to collapse and faithfully reform thescaffold's structure in a stimulus-responsive manner. A combination ofmechanical compression and dehydration is sufficient to compress thescaffolds developed in this work, allowing minimally invasive deliverythrough a conventional-gauge needle.

These results described herein demonstrated that shape-definedmacroporous alginate-based scaffolds were prepared with differentgeometric sizes and shapes, and successfully passed through a surgicalneedle without mechanical fracture, and all scaffolds regained theirthree-dimensional shape immediately (<1 s) after rehydration (FIG. 1).The fabrication method is capable to manufacture biocompatible,biodegradable and complicated macroporous tissue scaffolds efficientlyand economically. In addition to the application described herein, shapememory scaffolds are especially useful in applications in which large,structurally defined implants are required.

Example 2: Structural Integrity of Injectable Macroscopic Shape-DefinedGels

The deformation of conventional (nanoporous) and macroporous 1%MA-alginate gels under mechanical compression associated with shearforces was examined. Subject to mechanical compression, the gelsexperience a body of force, which results in a shape change. Theinfluence of the macropores on the gel mechanical properties was alsoevaluated since the stiffness of the scaffold dictate the extent of thedeformation under an applied shear force. Conventional gels give aYoung's modulus (i.e., the slope of the initial part of the stress vs.strain curves in FIG. 2) of 42±4 kPa in compression test. However,macroporous gels led to a dramatic reduction in the modulus to 4±2 kPa.As shown in FIG. 2, cylindrical (4 mm diameter×8 mm height) nanoporousgels reduced their heights by ˜16% when subjected to a vertical loadbefore mechanical fracture. In comparison, cylindrical macroporous gelsgive much larger deformation under lower mechanical stress, due to itslower modulus. Macroporous scaffolds attained 90% or more of compressionstrain without mechanical fracture, demonstrating their ability tomaintain their structural integrity after compression, compaction, andminimally invasive delivery. Also, these results confirmed that thescaffolds displayed shape memory in vitro.

In the hydrogels described herein, the large volume change of themacroporous shape-defined gels was caused by reversible collapse of theinterconnected pores. The collapsing pores force water contained in themacropores to flow out of the gel. Gel deformation and water convectionenhances water transport in and out of the gel. Once the mechanical loadis removed, the elastically deformed gel immediately returns to itsoriginal, undeformed shape-defined configuration in less than Is, assurrounding water was reabsorbed into the gel.

Example 3: Shape Memory Injectable Scaffolds As a Controlled DrugDelivery Carrier

Covalently crosslinked alginate scaffolds possessing shape memoryproperties were successfully used as a drug delivery system in vivo. Thegels having a predefined size and structure were able to exceptionallymaintain their structural features after minimally invasivesubcutaneously insertion in mice. Suspended gels in PBS werespontaneously hydrated with full geometric restoration after one singleinjection per site on the lower back of mice. Injected animals did notdemonstrate abnormalities in feeding, grooming, or behavior during thetime frame of the experiment, nor did they exhibit signs of distress.

The hydrogels maintained their hydrogel shape integrity at the site ofinjection. Animal studies performed to examine the integration of thespongy-like gels with the host tissue showed that the alginate-basedscaffolds were biocompatible and did not elicit an immune response orrejection when injected in mice. After 3 days post-injection,rhodamine-labeled scaffolds were surgically removed from mice andanalyzed. As shown in FIG. 3B, the scaffold guided in vivo tissueformation around the scaffold indicating the scaffolds could supporttissue growth and integration. Furthermore, fluorescent microscopy usedto visualize the rhodamine-labeled scaffold, noticeably displayed theoriginal geometry, structural integrity, square-defined shape retentionof the gels in vivo (FIG. 3C).

Rhodamine-labeled BSA was also used as a drug delivery model. Byproviding a drug depot at the site of injection, such devices achievehigh local drug concentrations without significant systemicadministration. Sustained release of BSA was achieved from the injectedsquare-defined scaffolds as shown in FIG. 3D. Targeted and controlleddelivery of rhodamine-labeled BSA in mice was quantified via real-timenon-invasive live imaging (FIG. 3A). Exemplary compound, BSA, was eitherphysically entrapped or chemically grafted to the scaffold during thecryopolymerization process. As illustrated in FIG. 3E, sustainedcontrolled release of BSA was achieved over of period of 4 months.Surprisingly, the release profiles for both types of BSA were similarindicating that the release is mainly mediated by matrix degradationover protein diffusion.

Example 4: Cryogel Compositions Enhance Survivability and LimitMigration of Injected Cells In Vivo

One application for the compositions and methods described herein is thenon-invasive method of cell injection based on cell-scaffoldintegration. Cell transplantation is a therapeutic option for patientswith impaired regional or global function due to cell death. However,the limited number of transplantation methods of cells is considered amajor factor limiting the efficacy of cell therapies. As cell andbioactive molecule carriers, injectable preformed scaffolds offer thepossibility of homogeneously distributing cells and molecular signalsthroughout the scaffold. Moreover, the scaffolds are injected directlyinto tissues or cavities, e.g., muscle, bone, skin, fat, organs, even ofirregular shape and size, in a minimally invasive manner. Thecompositions and methods described herein offer significant advantagessuch as injectability and efficient cell encapsulationpost-polymerization while allowing sufficient mechanical strength towithstand biomechanical loading and providing temporary support for thecells.

Square-shaped rhodamine-labeled RGD-containing alginate cryogels (4×4×1;units: mm) were prepared, purified, sterilized, and subsequently seededwith bioluminescent B16 cells, and maintained in culture for 6 hr incell culture medium before animal subcutaneous injection to promotecell-scaffold integration (FIGS. 4A, 4B, and 4C). Large interconnectedpores significantly enhanced cell seeding and distribution, whilemaintaining relatively high seeding efficiencies (>50%) and viability(>95%). To image bioluminescence of seeded B16 melanoma cells in vitro,0.15 mg/g of luciferin was added on top of the gel, which freelydiffused through the gel network, staining the cells and indicatinghomogeneous infiltration and depth viability of cells throughout the 3-Dconstruct (FIG. 4B). This is due to the effective nutrient delivery intoand waste removal from the inner regions of the scaffold. SEM imagesconfirmed a homogeneous distribution and engraftment of cells within thescaffold (FIG. 4C).

A unique characteristic of these cell/scaffold constructs is that whenan appropriate shear stress is applied, the deformable hydrogel isdramatically and reversibly compressed (up to 90% of its volume)resulting in injectable macroporous preformed scaffolds. This propertyallows gel/cell constructs to be delivered via syringe with highprecision to target sites. Homogenous cellular distribution and cellviability are unaffected by the shear thinning process and gel/cellconstructs stay fixed at the point of introduction, suggesting thatthese gels are useful for the delivery of cells to target biologicalsites in tissue regeneration efforts.

Subsequently, healthy C57BL/6 mice received a subcutaneous injection ontheir backs of 200×10³ B16's integrated into alginate macroporousscaffolds. The resulting injected gels were delivered to a targeted sitewhere they quickly recovered to their original mechanical rigidity withlocation permanency. As shown in FIG. 4D, cell-loaded rhodamine-labeledalginate scaffolds were syringe-delivered (1 cc, 16 G) with highprecision in the back of mice and visualized by in vivo optical liveimaging. Integration of melanoma B16 cells to RGD-modified alginatecryogel scaffolds and their injections into healthy mice wasinvestigated to demonstrate successful syringe-delivery and function ofpre-cultured cells while promoting homing, survival, and engraftment oftumorigenic cells. The results presented herein demonstrate that thedesigned tissue-engineered scaffolds mimic the natural environment wherecells normally reside, and as a result tumors are formed after everyinjection of tumorigenic cell-embedded matrix in healthy BALB/c mice.The inoculation of melanoma cells subcutaneously was monitored viareal-time non-invasive live imaging (FIG. 4D). The incidence of tumorformation and tumor growth was examined over a period of 9 days. Thesuccess of the melanoma B16 tumor model is clearly evident as shown inFIGS. 4D-4E. As an in vivo model, the cell/scaffold construct hasfulfilled several criteria: successful syringe-delivery with precisionto a target site and cell survival in their current local environmentresulting in tumor formation.

As described herein rhodamine-labeled (1) and rhodamine-labeledRGD-modified (2) cell-seeded alginate cryogels were administered in miceto study the effect of cell-engraftment in cell transplantation andhoming. As a control, a bolus of free cells (B) was also injected.Rhodamine-labeled scaffolds were successfully injected subcutaneously asshown in FIG. 4D. Except for the bolus injection site, red-emittingrhodamine dyes show intense fluorescent red spots in each side of themice's back indicating in vivo localization of cell-seeded scaffolds.After 2 days post-injection, bioluminescence of cell-seeded scaffoldswas measured 30 min after intraperitoneal injection of luciferin. Asshown in FIG. 4E, bioluminescence for injected RGD-modified cell-seededgels was particularly brighter when compared to the plain scaffoldsshowing the necessity to incorporate RGD to the polymeric network tosupport cell-engraftment and thus efficient cell transplantation. Forthe injection of the cellular bolus, the absence of bioluminescencesuggests minimal cell retention at the injection site, rapid cellmigration, and likely limited cell transplants survival. Similarly, 9days post-injection, bioluminescence of cell-seeded scaffolds was mainlyapparent for RGD-modified scaffolds confirming the developednon-invasive method for cell injection based on cell-scaffoldintegration is crucial to decrease migration, promote homing, enhancesurvivability, and engraftment of cells in vivo (FIG. 4F).

Decreasing the rapid cell death that occurs within a few days aftertransplantation of graft cells is of great relevance for the success ofcell transplantation therapies. The results presented herein confirmthat the incorporation of the cell-adhesive peptide plays a key role inregulating interactions between cells and the scaffold and cell-fate.These gels are also suitable for use as a delivery system for thesustained delivery of proteins (e.g., growth factors) involved in celldifferentiation and maturation (FIG. 3E). This technique is also a toolfor enhancing stem cell survival in vivo.

Example 5: Injectable Biodegradable Cryogels for ImmunotherapyApplications

A minimally invasive scaffold-based active vaccine containing hostpathogens was developed for the therapeutic treatment of cancer. In thecase of cancer, the immune system needs an external boost fromimmunotherapies to be able to become more effective in fighting cancer.The active immunotherapy system described herein was designed tostimulate the patient's immune system, with the objective of promotingan antigen-specific antitumor effect using the body's own immune cells.In addition, the cryogel-vaccine leads to a durable antitumor responsethat protects tumor recurrence. Dendritic cells (DCs) areantigen-presenting cells critically involved in regulating the immunesystem. The vaccine mediates in situ manipulation of dendritic cellrecruitment, activation, and their dispersion to the lymph nodes.Cytosine-guanosine oligonucleotide (CpG-ODN) was used as an adjuvantfurther stimulate responses to the vaccine.

As shown in FIG. 5A, both components (adjuvant and cytokine) can beeasily incorporated into the cryogel matrix and released in a sustainedfashion to recruit and host DCs, and subsequently present cancerantigens from the irradiated cells (or other cell-associated antigens)and danger signals to activate resident naïve DCs and promote theirhoming to the lymph nodes, which is necessary for a robust anti-cancerimmune response. Specific and protective anti-tumor immunity wasgenerated with our minimally invasive alginate-based active vaccine, as80% survival was achieved in animals that otherwise die from cancerwithin a couple of months. The data using the cryogel-based prophylacticvaccine for melanoma was shown to induce a very strong immunologicmemory, as 100% survival was achieved in the rechallenged animalsfollowing 100 days post vaccination.

Different tumor cell-associated antigens are used in the cellularcryogel-based vaccine platform, thereby permitting treatment orprophylaxis for a variety of cancers. Active specific immunotherapyinvolves the priming of the immune system in order to generate a T-cellresponse against tumor-associated antigens. One example of the activespecific approach is adoptive T-cell therapy, which involves the ex vivocultivation of T cells with demonstrated activity against a specifictarget cancer antigen. Cells are obtained from the subject, purified,and cultured. Such ex vivo cultivation increases the frequency of theseT cells to achieve therapeutic levels. The cells are then infused backinto the patient via injectable alginate-based cryogel.

Creating an infection-mimicking microenvironment by appropriatelypresenting exogenous cytokines (e.g., GM-CSF) and danger signals (e.g.,CpG-ODN), in concert with cancer antigen provides a means to preciselycontrol the number and timing of DC tr afficking and activation, insitu. At different time points post scaffold-based vaccine injection(vax C), cells were isolated from the cryogels and surrounding tissues,spleen, and lymph nodes (LN) for cell counting andfluorescence-activated cell sorting (FACS) analysis to determine theoverall number of cells and percentage of DCs (CD11c+ cells) and T cells(CD3+ cells). Cells infiltrating the vaccine site and the enlargement ofspleen and LN after vaccination revealed a significant immunologicresponse to cancer. The increased numbers of immune system cellsfighting cancer antigens made the two organs expand and become“swollen.” As shown in FIG. 3A, the total numbers of cells increaseddramatically for the vaccinated (V) and vaccinated/challenged (VC) micewhen compared to the control groups (C) for the spleen, LN, andcryogels. The increase number of cells remained relatively high withinthe first 2 weeks post vaccination and started to noticeably drop by day13 impaired with a reduction of immunologic and inflammatory responses.

Macroporous cryogel matrices were fabricated for controlled release ofGM-CSF to recruit and house host DCs, and with an interconnected porousstructure that allows for cell infiltration and subsequently presentcancer antigens (irradiated B16F10 melanoma cells) and danger signals(CpG-ODN) to activate the resident DCs and dramatically enhance theirhoming to lymph nodes and proliferation. Matrices were loaded with 3 mgof GM-CSF and injected into the subcutaneous pockets of C57BL/6J mice.FIG. 3B indicates that the cryogel vaccine controls or therapeuticallyalters immune cell trafficking and activation in the body. Within thefirst 10 d post vaccination, a large number of DCs are recruited to thevaccine site. As these activated DCs may home to the inguinal lymphnodes and spleen, present antigens to naïve T cells, and stimulate andexpand specific T-cell populations that elicit anti-tumor responses, thetotal number of CD11c(+) DCs is inversely proportional to the totalnumber of CD3(+) T cells. FACS analysis of cells infiltrating thevaccine site revealed a significant CD3(+) T cell response peaking atday 13. Local CD3(+) T cell numbers dropped sharply by day 24 and werenegligible at day 30.

These cryogel matrices released approximately 20% of their bioactiveGM-CSF load within the first 5 days, followed by slow and sustainedrelease of bioactive GM-CSF over the next 10 days (FIG. 8, cryogel A);this release profile was chosen to allow diffusion of the factor throughthe surrounding tissue to effectively recruit resident DCs. Cryogels canbe successfully used for specific spatiotemporal delivery of severaldrugs, as the incorporation of a second biomolecule (CpG-ODN) did notalter the release profile of GM-CSF over time (FIG. 8, cryogel B).However, slowly degrading PLG microspheres integrated in the scaffoldsseem to release GM-CSF much more slowly than pure cryogels (5% vs 24%release at day 14). Hybrid cryogel have been created as a potentialcarrier for controlled delivery of hydrophobic and/or low moleculeweight drugs. Our results not only provide a new strategy for deliverydrugs from an injectable 3-D preformed macroporous scaffolds as asustained-release drug carrier but also open an avenue for the design ofnew hybrid injectable hydrogels.

Example 6: Injectable Biodegradable Cryogels as a Gene Delivery System

Nonviral gene delivery systems based upon polycation/plasmid DNAcomplexes are gaining recognition as an alternative to viral genevectors for their potential in avoiding immunogenicity and toxicityproblems inherent in viral systems. Studies were carried out todetermine the feasibility of using a controlled release system based onencapsulated condensed plasmid DNA in injectable cryogels to achievegene transfer in the surrounding tissues after injection. A uniquefeature of the cryogel-based gene delivery system is thebiodegradability of the polymeric system, which can provide a sustainedrelease of DNA at different rates depending on the polymer, cross-linkdensity, mass fraction, and porosity created during the cryogelationprocess. Encapsulated DNA complexed with polyethylenimine (PEI), anondegradable cationic polymer known to be an effective gene carrier,and naked PEI/DNA complexes, which were prepared at a ratio of 7:1(PEI:DNA) were injected subcutaneously on the lower back of naïve miceusing luciferase as a reporter gene (FIG. 9). At 1 day after injection,encapsulated PEI/DNA displayed strong bioluminescence providing thehighest transgene expression at −10 photons/s, about two-order ofmagnitude higher than that produced by naked PEI/DNA. After 10 days, theexpression levels for naked PEI/DNA were about the same as day 1 butincreased by 1 order of magnitude when released in a controllablefashion from the cryogels. Till 29 days, encapsulated PEI/DNA stillprovided a level of transgene expression at ˜10⁷ photons/s, similar tothat observed at previous time points. This level was significantlyhigher than those offered by naked PEI/DNA.

In this study, subcutaneous gene delivery allowed gene expression on thelower back of naïve mice, although the distribution pattern andintensity was vehicle-dependent. Naked PEI/DNA complexes producedlimited bioluminescence (signal nearly above background), probablybecause of its vulnerability to DNAses. However, encapsulated PEI/DNAcomplexes in cryogels used in this study provided a targeted andsustained high level of gene expression around the injection site for atleast 3 weeks. These findings indicate that a 3-D macroporous scaffoldmay facilitate sustained release and efficient cell transfection ofpolymer/DNA complexes.

In summary, the present approach has demonstrated that cryogels promotegene transfection to surrounding cells in the subcutis of mice, with anefficiency superior in terms of prolonged gene expression to naked DNA.The results establish an injectable delivery system as an effective genecarrier applicable to program or treat targeted cells.

Example 7: Immunologically Active Injectable Sponges

In this example, a novel cell-based immunotherapy for cancer wasdeveloped with the aim of enhancing anti-tumor immunity, reducing exvivo cell manipulation, preventing repetitive vaccination, andminimizing invasiveness during administration. For this cell-basedimmunotherapy, a minimally invasive sponge-based vaccine was designed,containing living irradiated tumor cells along with immunomodulatoryfactors for the prophylactic treatment of melanoma. In the framework ofscaffold-based cancer immunotherapy, macroporous 3D alginate cryogelswere prepared using MA-alginate by the process of cryopolymerization at−20° C. using a free-radical cross-linking mechanism. During cryotropicgelation, most of the solvent (water) was frozen, and the dissolvedsolutes (macromonomers, immunomodulator agents, and initiator system)were concentrated in small semifrozen regions called nonfrozen liquidmicrophase, in which the free-radical cryopolymerization and gelationproceeded with time (FIG. 5A). After complete polymerization, and whensubsequently incubated at room temperature (RT) and washed with water toremove unreacted polymeric precursors, the ice crystals melted and leftbehind a system of large, continuously interconnected macroporesthroughout the entire cryogel construct (FIGS. 5B-C). Irradiated tumorcells (3500 rads) were initially seeded in sterilized cryogels (FIG.5A), resulting in cells being homogeneously distributed in gel pores dueto the unique interconnected macroporous network. Cryogels were furthermodified with RGD peptides to enhance tumor cell attachment by way ofintegrin-mediated cell adhesion motifs for cell-surface antigen display.RGD modification of alginate cryogels enhanced attachment and spreadingof cells after 6 h incubation prior to vaccination (FIG. 5D). Autologoustumor cells are a source of tumor-associated antigens (TAA) forvaccination purposes, since, by definition, all relevant candidate TAAshould be contained within them. A unique feature of these cryogels isthat when an appropriate mechanical force is applied, the gel willshear-collapse, resulting in a biomaterial that flows through aconventional-gauge needle. Unlike traditional nanoporous hydrogels,which are rather brittle, MA-alginate cryogels are elastic, soft,sponge-like materials that can withstand large deformations and can beeasily compressed to a fraction of their sizes without beingmechanically damaged. See Bencherif S A et al. Proc. Natl. Acad. Sci.USA. 2012; 109:19590-5. Shape-defined macroporous alginate-basedscaffold vaccines prepared with a square-shape size were successfullypassed through a surgical needle without mechanical fracture or celldispersion. See id. After the shear force is removed, the scaffoldsquickly recovered their original shapes once placed subcutaneously(FIGS. 14A-B).

Macroporous alginate sponges were designed to present GM-CSF, dangersignals, and cancer antigens in a defined spatiotemporal manner in vivoand serve to as a residence for recruited dendritic cells as they areprogrammed. GM-CSF was physically encapsulated (85% efficiency) intocryogels through the crosslinking process. Cryogels were successfullyused to control the spatiotemporal delivery of several biomolecules(adjuvant and cytokine), as the incorporation of a secondimmunostimulatory agent (CpG-ODN) did not alter the release profile ofGM-CSF over time (FIG. 15B), although the encapsulation efficiency wasslightly lower (75%). Similarly to GM-CSF, TLR-activating CpG-ODN wasphysically immobilized within the polymer network of alginate and theencapsulation efficiency (approximately 45%) was independent of theincorporation of GM-CSF (FIG. 15C). The inferior encapsulationefficiency of negatively charged CpG-ODN could be attributed to acombination of its low molecular mass coupled with electrostaticrepulsions due to negatively charged alginate chains. These matricesreleased approximately 80% of their bioactive GM-CSF and CpG-ODN loadswithin the first 4 days, followed by slow and sustained release of thebioactive immunostimulatory factors over the next month (FIGS. 15B-D);these release profiles were chosen to enable diffusion of the factorsthrough the surrounding tissue to effectively recruit and activateresident dendritic cells.

Next, experiments were performed to determine whether the cryogelvaccines were immunologically active. Following vaccination, a localprogressive swelling at the vaccination injection site was observedwithin the first two weeks, which is indicative of a strong inflammatoryresponse and recruitment of immune cells (FIG. 16). As DC are keyplayers in the defense against cancer because of their role in T cellpriming and T helper differentiation, changes in their phenotype uponexposure to CpG-ODN-loaded cryogels in vitro were detected. To this end,isolated bone marrow-derived DC (BMDC) (FIG. 17A) that were 97% viable(FIG. 18) and with at least 90% of cells staining positive for CD11cwere exposed to cryogels or conditioned media (FIG. 17B). Approximately60% of BMDC (group C, FIG. 17C) exposed to CpG-loaded cryogels stainedpositive for CD86 and MHCII, similarly to the positive control (group D,FIG. 17C) of BMDC cultured in medium supplemented with CpG-ODN. However,similar to the negative control (group A, FIG. 3C), the cells (group B,FIG. 17C) cultured with blank cryogels did not activate BMDC. Inaddition, the upregulation of interleukin (IL)-12 secretion by BMDC wasinvestigated. Activated DC are known to generate signals, such as theIL-12 cytokine, that alert the immune system to potentially dangerousmaterial and modulate subsequent lymphocyte activation anddifferentiation. The stimulation from released CpG-ODN inducedactivation of immature BMDC, as indicated by a significant increase ofIL-12 production (233 pg/mL). This is in contrast to the adjuvant-freecryogels, which were not immunogenic and led to a low concentration ofIL-12 (9 pg/mL). See FIG. 17D. This data shows that the cryogels are notonly able to release CpG-ODN, but are immunogenic since they have thepotential to activate resident DC within and around the matrix.

To check the capacity of the cryogel sponges to promote cellularinfiltration in situ, blank cryogels and their nanoporous counterpartswere implanted into the subcutaneous pockets of C57BL/6J mice. After 1day, the scaffolds were explanted and the total number of infiltratedcells quantified. Cryogels facilitated substantial cellular infiltrationwhen compared with nanoporous hydrogels, as a total number of 6×10⁶cells were recruited to the cryogels as opposed to 0.2×10⁶ cellsrecruited to nanoporous hydrogels, representing an increase of 2900%.Cryogels have an interconnected macroporous architecture, which isadvantageous over nanoporous hydrogel scaffolds with respect to theirability to facilitate cellular infiltration and trafficking.

Further, selective cell recruitment and spatially controlled cell homingwas tested. The ability of the cryogels to selectively recruit and homeDC at the vaccination site was tested. GM-CSF loaded cryogels were usedto provide a 3-D matrix in the surrounding tissue and regulatedendritic-cell recruitment for their activation and subsequentdispersion (FIG. 19A). Since 80% of GM-CSF was released from thescaffolds within the first few days following injection, the capacity ofthe cryogel vaccines to recruit and host DC after only one daypost-injection was determined. Cryogel sponges loaded with 1.5 μg ofGM-CSF were injected into the subcutaneous pockets of C57BL/6J mice.Histological analysis at day 1 revealed that the total cellularinfiltration into cryogels (FIGS. 19B and 19E) was significantlyenhanced compared with the control (no GM-CSF incorporated; FIG. 19B-C).Fluorescence-activated cell sorting (FACS) analysis for CD11b⁺ CD11c⁺ DCshowed that GM-CSF increased not only the total resident cell number incomparison to blank cryogels, but also the percentage of cells that weredendritic cells (FIGS. 19C-E and 20). Surprisingly, the number ofdendritic cells residing in the material as a result of GM-CSF deliverywas 3 times higher than the number of dendritic cells that are commonlyprogrammed and administered by ex vivo protocols (˜10⁶ cells). GM-CSFdelivery promoted greater cellular penetration into the cryogel sponges,as indicated by the measurement of DC numbers (FIG. 19D) andhistological analysis (FIGS. 19D-E), thereby allowing for the subsequentprogramming of resident DC precursors and DC.

Example 8: Autologous Tumor Cell Vaccines

Creating an infection-mimicking microenvironment by appropriatelypresenting exogenous cytokines (e.g., GM-CSF) and danger signals (e.g.,CpG-ODN) in concert with antigen-displaying cells may provide an avenueto precisely control the number and timing of DC trafficking andactivation in situ—this example describes experiments to test thisconcept. At different time points post scaffold-based vaccine injection,cells were isolated from the cryogels and from the surrounding tissues,spleen, and lymph nodes (LN) for cell counting andfluorescence-activated cell sorting (FACS) analysis in order todetermine the overall number of cells and percentage of DC (CD11c⁺cells) and T cells (CD3⁺ cells) (FIGS. 7A-B). A significant immunologicresponse to the vaccines was reflected in the number of cellsinfiltrating the vaccine site along with the enlargement of the spleenand lymph node (FIG. 21). The total numbers of cells increaseddramatically for the vaccinated (V) and vaccinated/challenged (VC) micewhen compared to the control groups (C) for the spleen, LN, and cryogels(FIG. 7A). The increased number of cells remained relatively high withinthe first 2 weeks following vaccination and started to noticeably dropby day 13.

As described previously, macroporous cryogel matrices were fabricatedfor controlled release of GM-CSF to recruit and house host DC. Theinterconnected porous structure that allows for cell infiltration andthe presentation of cancer antigens (irradiated B16F10 melanoma cells)and danger signals (CpG-ODN) that activates the resident DC anddramatically enhances their proliferation and homing to lymph nodes.FIGS. 7A-B show that the cryogel vaccine was able to control immune celltrafficking and activation in the body. Within the first 2 weeks postvaccination, a large number of DC were recruited to the vaccine site(FIG. 7B). These activated DC could then home to the inguinal lymphnodes and spleen, present antigens to naïve T cells, and stimulate andexpand specific T-cell populations that elicit anti-tumor responses.CD11c⁺ DC were rapidly and massively recruited and housed at the vaccinesite, peaking at day 9, indicating that this cryogel vaccine provides asupportive microenvironment for cell-trafficking during the immuneresponse. Later, DC were subsequently released over time while beingsubstituted with CD3⁺ T cells (FIG. 7B). FACS analysis of cellsinfiltrating the vaccine site revealed a significant CD3⁺ T cellmigration peaking at day 13. Local CD3⁺ T cell numbers dropped sharplyby day 24 and were negligible at day 30, likely due to antigen clearance(FIG. 7B).

Example 9: Cryogel Vaccines Promote CD8+DC, Plasmacytoid DC and CD8+ TCells while Reducing FoxP3+ Cell Numbers

In order to better understand the cellular response to the cryogelvaccines, the DC and T cell types at the vaccine site, draining lymphnodes, and spleen were examined at the peak of cell infiltration alongwith cytokine expression at the vaccine site (FIGS. 22A-G). Similarly tothe examples above, three different conditions were tested: blankcryogels (C: control), cryogel vaccines with CpG-ODN/GM-CSF (V:vaccinated mice), and cryogel vaccines with CpG-ODN/GM-CSF challengedwith tumor cells six days later (VC: vaccinated/challenged). Analysiswas performed at three sites: cryogel localization, draining lymphnodes, and spleen. Nine days following vaccination, the number of CD11c+cells as well as the number of plasmacytoid and CD8+DC in the vaccinatedgroups at the implantation site and the draining lymph node were greaterthan that for the blank cryogel group (FIGS. 22A-B). In the spleen, themean number of DC, plasmacytoid DC, and CD8+DC for the vaccinatedconditions was higher, but the difference does not achieve statisticalsignificance (FIG. 22C). In the lymph node, there was a greater numberof plasmacytoid DC in the vaccinated/challenged group in comparison tothe vaccinated group, otherwise the number of DC were similar for bothgroups at the different sites. For both of the vaccinated groups at allof the sites examined, plasmacytoid DC represented greater than 50% ofall of the DC while CD8+DC constituted a quarter to a third of theremaining DC. This set of data demonstrates that these cancer vaccinesengineered to selectively trigger Toll-like receptors 9 have thepotential to lead to increased immunogenicity.

At day 13, the number of CD3+ T lymphocytes in the cryogel sponges andthe lymph nodes was greater in the vaccinated animals compared to theanimals that received only blank cryogel injections (FIG. 22D). As shownin FIG. 22E, the number of CD8+ T cells was greater in the vaccinatedand vaccinated/challenged mice in comparison to the blank controlcryogels at all of the investigated sites, however there was nostatistical difference between the vaccinated and vaccinated/challengedgroups. These data demonstrate that an immune response is triggeredafter vaccination.

Further, the potency of the cancer vaccine has been associated with theelimination of different metabolic pathways in tumor-associated immunesuppression via reduction of the impacts of regulatory T cells (Tregs).Tregs, identified as the FoxP3+ subset of CD4+ T cells, play pivotalroles in controlling the balance between immune stimulation andsuppression. The ratio of CD8+ effectors to FoxP3+ T cells at day 23,however, was greater in the vaccinated mice in comparison to the blankcontrols at all of the sites and in the lymph nodes and cryogels for thevaccinated/challenged mice (FIG. 22F). For the vaccinated animals, inthe cryogel sponge, the ratio of CD8+ T cells to FoxP3+ T cells was over2.5 times greater than in blank cryogels. Overall, there was nosignificant difference in T cell numbers between the vaccinated and thevaccinated/challenged mice. FoxP3+ Tregs impose critical barriers thatwere overcome naturally via infection-mimicking cryogel vaccines,allowing the priming of protective antigen-specific CD8+ T cells.

Additionally, at day 13, the cryogels and surrounding tissue for thethree conditions were resected, and several biomarkers were measured(FIG. 22G). The concentrations of RANTES, eotaxin, IL-1β, IL-1α, GM-CSF,IL-10, MCP-1α, and MCP-1β were greater in the tissue of the vaccinatedanimals compared to the animals that received blank cryogels. Inparticular, the concentration of RANTES (CCL5), a cytokine that plays anactive role in recruiting leukocytes into inflammatory sites, wasmarkedly elevated and found to be at least 10-fold higher. Further, theproduction of a unique combination of cytokines, such as eotaxin, IL-1β,and GM-CSF, were quantified to be over 5 fold greater. Similarly, theconcentration of IL-1β, GM-CSF, and MCP-1β biomarkers was greater in thevaccine/challenged group in comparison to the blank control group. Thesedata demonstrate that the biomarker profiles are affected (to varyingextents) when vaccinated mice are exposed to tumor cells duringchallenge. The concentration of IL-12, TNF-α, and INF-α were similaramong the groups. There was no difference in the concentrations ofcytokines between the vaccinated and the vaccinated/challenged groups.

Example 10: Activity of Cryogel Vaccines in Melanoma Model

The ability of the vaccine to evoke anti-tumor immunity was next testedin the prophylactic setting. GM-CSF and CpG-ODN can be released in asustained fashion to recruit and host DC, and subsequently presentcancer antigens from the irradiated cells and danger signals to activateresident naïve DC and promote their homing to the lymph nodes, which isnecessary for a robust anti-cancer immune response. Specific andprotective anti-tumor immunity was generated with the minimally invasivealginate-based active vaccine, as 80% survival was achieved in animalsthat otherwise die from cancer within a couple of months (FIG. 23A).This cryogel-based prophylactic vaccine for melanoma induced a strongimmunologic memory, as 100% survival was achieved in the rechallengedanimals following 126 days post vaccination (FIG. 23C). Thecryogel-based vaccines provided a powerful short-term and long-termprotective immunity when antigen-displaying cells, adjuvant, andchemoattractant were all combined together. The combination ofCpG-loaded cryogel and genetically engineered GM-CSF-secreting tumorcells resulted in synergistic enhancement of cellular-vaccine efficacyin vivo. The induced effect was comparable to that of thecryogel-vaccines during the first tumor challenge. However,cryogel-vaccine performance was more effective during the second tumorchallenge; this demonstrates that encapsulating GM-CSF within thepolymer scaffold can provide a more suitable spatio-temporal release ofcytokines, resulting in long-term active immunological memory. Inaddition, the benefit of providing a 3D matrix for the housing ofrecruited dendritic cells while they are programmed was demonstrated bythe failure of long-term protection of bolus vaccine formulationsconsisting of bolus injections of irradiated tumor cells, with andwithout CpG-ODN (FIG. 23C). The combination of tumor antigen-displayingB16-F10 cells, GM-CSF, and TLR9-activating CpG-ODN in the vaccine matrixwas required for optimal tumor protection as a markedly enhanced mousesurvival was achieved following two tumor challenges (FIG. 23D).

The ability of continuous dendritic cell recruitment and programming togenerate an immune response was next tested in the melanoma model. Thescaffold-based vaccines provided significant protection, especially whenGM-CSF was released from the device. Animal survival increased from 30%to 80% when mice were vaccinated with sponges loaded with GM-CSFtransduced tumor cells as opposed to regular tumor cells (FIG. 23A).This infection-mimicking material induced better immune protection thanthat obtained with previously reported cell-based therapies. See DranoffG et al. Proc. Natl. Acad. Sci. USA. 1993; 90:3539-43; and Dranoff G.Immunol. Rev. 2002; 188:147-54. Materials presenting CpG-ODN or GM-CSFalone with tumor cells resulted in only a 70% and 10% survival duringthe first tumor challenge, respectively, and only a 50% overall survivalduring the second tumor challenge for the CpG-ODN loaded cryogelvaccine, indicating the benefit of recruiting dendritic cells withGM-CSF for long-term protection (FIGS. 23C-D). Except for theGM-CSF-secreting B16-F10 cells vaccine, mice immunized with thedifferent regimens including TLR-activating CpG-ODN displayed delay intime of tumor appearance, significantly retarded tumor growth, andprolonged survival (FIGS. 23A-B). Particularly, vaccination of mice withthe cryogel vaccine containing both immunomodulators (GM-CSF andCpG-ODN) significantly decreased the rate of tumor progression (FIG.23B), and improved life expectancy over controls was observed (FIG.23A). In contrast, a single treatment with irradiated, GM-CSF-secretingB16-F10 cells, a cell therapy now in clinical trials, attenuated tumorgrowth similarly to CpG-ODN loaded cryogel vaccines; however, thesurvival rate was significantly reduced compared to vaccines containingboth immunomodulators, 30% vs. 80% (FIGS. 23A-B). Injecting tumorcell-loaded sponges without immunomodulatory factors resulted in littleimmune protection suggesting that the material without GM-CSF andCpG-ODN was inert to DC. However, injectable alginate sponges containingboth immunostimulatory factors and syngeneic irradiated tumor cellsprovided a suitable residence to recruit and program DC to elicit apotent immunity capable of preventing melanoma. To ensure that thevaccines are safe as well as effective, explanted implants and organswere sent for toxicological analysis. The pathology report indicated noevidence of pathologic changes, although minimal granulomas were locatedat the vaccine implants, suggesting long-term effectiveness. The absenceof toxicity to the liver, kidney, or other organs suggests that thescaffold-based vaccines have minimal adverse effects and no safetyconcerns.

Example 11: Cryogel Vaccines for Anti-Tumor Immunity Against BreastCancer

Several reports from experimental models and clinical studies havedescribed that HER-2/neu is an immunogenic molecule since it generatesantibody responses and activation of HER-2/neu peptide-specificcytotoxic T lymphocytes and T helpers. Therefore, here theHER-2/neu-specific humoral response was studied by investigatingHER-2/neu-specific IgG antibody production in response to theHER-2/neu-based cryogel breast cancer vaccine in BALB/c mice.

The presence of HER-2/neu-specific IgG antibody was assessed by flowcytometric analysis on CT26-Her-2/neu colon adenocarcinoma cellsincubated with mouse sera at different time points prior to challenge(days 14 and 28) and 1 week post-challenge (day 37), followed byfluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody. Thevaccines were found to be safe and effective in raising vaccinetype-dependent HER-2/neu immunity, as observed with HER-2/neu-specificIgG stimulation in immunized mice (groups 1, 2, 3, 4, and 5 of FIG. 24),indicating a neu-specific Th2-type response.

Throughout the course of the study, low HER-2/neu-specific antibodytiters were detected in the sera of unvaccinated mice (groups 7 and 8)or mice immunized with blank cryogels (group 6). Mice vaccinated withHER-2/neu positive breast cancer cells did not induce a strongneu-specific IgG antibody response at day 14. However, from day 28,cryogels with tumor cell-associated antigens (groups 2 and 3) resultedin an approximately 70-fold increase of HER-2/neu-specific IgG antibodywhen compared to controls (groups 5, 6, and 7) and up to a 7-foldincrease of HER-2/neu-specific IgG antibody when compared to cryogelswith tumor lysate-associated antigens (group 1) and to bolus injectionvaccines (groups 4 and 5) (FIG. 24).

Specific and protective anti-tumor immunity was generated with theseminimally invasive alginate-based active vaccines. As shown in FIG. 25,the cryogel-based prophylactic vaccines for breast cancer induced astrong humoral immunologic response. In particular, at day 78 (ongoingstudy), 80% and 100% survival was achieved in animals from groups 2 and3, respectively, that otherwise would die from cancer within a month(groups 6 and 7). Bolus injections of vaccine also promoted a robustanti-HER-2/neu immune response, as 80% of mice (groups 4 and 5) werestill alive at day 78 following animal challenge (FIG. 25). However,cryogel vaccines with tumor lysate-associated antigens induced a limitedprotective anti-HER2/neu immune response, as the mice died within 2months.

Taken together, this unique and flexible cryogel vaccine system is apromising cell-based immunotherapy approach for the treatment of breastcancer using allogeneic tumor-cells. The data presented hereindemonstrates that the HER-2/neu-based cryogel vaccine provides a morepotent prophylactic protection against mammary cancer than the positivecontrol (G-vax vaccine) in mice, which is in agreement with the higheranti-HER-2/neu antibody levels detected in the blood. This data showsthat the cryogel-based vaccine is over 80% effective. To test sustainedtumor immunity to prevent relapse, at day 100 following immunization,the mice will be rechallenged with 105 HER-2/neu-overexpressing breastcancer cells and monitored for the onset of tumor development.

Example 12: Bulk Cryogel Behavior

The bulk behavior of cryogels formed with GelMA macromonomerconcentrations between 1 and 2% (w/v) was studied to assess theirsuitability as injectable preformed hydrogels. 1.0% cryoGelMA showed asuperior interconnected porosity of 91±2%, relative to 1.5 and 2.0% gelswhich had mean interconnected porosities of less than 5% (FIG. 26C).Mechanical testing revealed that cryogels made with 1.5% and 2.0% GelMAled to brittle gels that could not undergo large strains (FIGS. 28A-C).However, 1.0% cryoGelMA gels demonstrated complete shape recovery whenreleased after deformation to high strains (FIG. 29A). 1.0% cryoGelMAgels collapsed dramatically when water was wicked away from theinterconnected pores, and rapidly reassumed their previous shape whenrehydrated (FIG. 29B). Due to their ability to undergo large deformationand allow rapid influx/efflux of water, the potential of 1.0% cryoGelMAgels to be injected through a conventional needle was next analyzed.Disc-shaped cryoGelMA gels (5 mm diameter, 2 mm thickness) couldcollapse, travel through a 16 G needle (1.65 mm inner diameter), andquickly (260±80 ms, n=10) return to their original geometry afterexiting the needle (FIG. 29C). Based on these findings, 1.0% cryoGelMAwas used in gelatin cryogel studies described herein.

Example 13: Gelatin Cryogel Scaffold Architecture

Since cell trafficking within scaffolds is subject to sufficient poresize (>10 μm) and interconnectivity, the microarchitecture of cryoGelMAgels was next assessed. Scanning electron microscopy (SEM) revealed ahighly porous surface and scaffold interior (FIG. 30A). Since theprocessing used to prepare the hydrogels for SEM can cause shrinkage,2-photon fluorescence imaging of rhodamine-cryoGelMA was used to studythe structure of these scaffolds in their hydrated state (FIG. 30B). Thegels demonstrated a highly interconnected pore structure, with the porediameter increasing with depth into the scaffold from the surface to theinterior, likely due to a temperature gradient within the scaffoldduring the freezing process. The hydrated pore size from the scaffoldsurface to a depth of 350 μm varied between 20-300 μm. Together thesedata indicate that cryoGelMA has an interconnected porous structure withpore sizes compatible with cell trafficking, movement, or migration.

Example 14: Gelatin Cryogel Cell Compatibility

The suitability of cryoGelMA gels as a substrate for cell attachment andgrowth was also tested. Free fluid was first wicked from cryoGelMA gels,and a cell suspension of NIH 3T3 fibroblasts, a cell line commonly usedfor cell compatibility testing, was then placed on the gels. 3T3 cellswere found distributed throughout the scaffold volume, as seen with2-photon microscopy (FIG. 31A). To study the interaction of cells withcryoGelMA, SEM was used to assess 3T3 cell morphology at the scaffoldsurface (FIG. 31B). 3T3 cells attached to the cryoGelMA surface andassumed their characteristic spindle-like morphology after 1 day ofculture. Subsequent culture for 2 days led to the formation of amonolayer of cells on the surface of the cryogel, and deposition ofextracellular matrix by the fibroblasts on the cryogel surface (FIGS.32A-B). Cell coverage of the cryogel surface was also seen to increaseover the culture period using fluorescence microscopy, and high cellviability was maintained on the scaffold surface (FIGS. 33A-B). Cryogelswere also seen to contract slightly over the 3 day culture period,indicating that 3T3 cells were exerting traction forces on the cryogel.Consistent with this observation, F-actin staining of histologicalsections showed actin stress fiber formation within cells in thescaffold interior (FIG. 31C).

Retrieval and analysis of cells from cryoGelMA scaffolds over theculture period showed maintenance of >90% cell viability (FIG. 31D).Cell number and metabolic activity increased over the culture period(FIG. 31D). Cell proliferation on cryoGelMA scaffolds was furtherstudied by characterizing new DNA synthesis. BrDU incorporation after 1d of culture showed cells were proliferating on cryoGelMA gels, albeitat a lower rate than cells cultured on tissue culture polystyrene (FIG.34), as is expected when a high density of cells is cultured in a 3Denvironment relative to a sparse 2D culture.

To assess whether proliferation occurred uniformly on cryoGelMAscaffolds, histological sections were stained after pulsing cell-ladenscaffolds with EdU (FIG. 31E). EdU-positive nuclei could be seenthroughout the scaffold thickness. Collectively, these results indicatethat cryoGelMA provides a substrate for cell attachment, sustaining cellviability, and allowing cell proliferation.

Example 15: Enzymatic Degradation of Gelatin Cryogels In Vitro

Studies were also performed to test whether modification of gelatin withmethacrylate groups and subsequent cryopolymerization preserved theinherent enzymatic degradability of gelatin. Fluorescent cryogels werefirst incubated in the presence of 25 U/ml collagenase type II, andfluorescence in the supernatant was monitored as a proxy for geldegradation (FIG. 35A). This analysis revealed that cryoGelMA gels couldbe degraded completely in the presence of collagenase over a period of10 days.

The ability of the mammalian gelatinases, MMP-2 and -9, to degradegelatin with a high degree of methacrylation was next assessed. GelMAwas incorporated as a substrate into a polyacrylamide gel and zymographywas performed with recombinant mouse and human gelatinases (FIG. 35B).After Coomassie staining of the gel, bands of GelMA degradation wereobserved with all of the enzymes tested, indicating preservation ofenzymatic degradability following methacrylation of gelatin. Short-termdegradation studies of rhodamine-cryoGelMA gels by activated mammaliangelatinases were also conducted (FIG. 35C). All of the enzymes testedwere seen to accelerate gel degradation compared to buffer alone.Together these results demonstrate that cryoGelMA is enzymaticallydegraded by collagenase and mammalian gelatinases.

Example 16: In Vivo Injection of Gelatin Cryogels

The in vivo response to cryoGelMA scaffolds was also explored. CryoGelMAgels injected subcutaneously through a conventional needle regainedtheir original shape under the skin (FIGS. 36A-B). Implanted scaffoldswere covered by a thin fibrous capsule after 2 months and underwent areduction in bulk size. H&E staining revealed mild inflammation at thescaffold border after 1 week with the presence of mononuclear phagocytesand lymphocytes and sparse infiltration of the scaffold by mononuclearcells (FIG. 36C). At 2 months post-implant, a foreign body reaction waspresent at the scaffold border with the presence of macrophages,multinucleated giant cells, fibroblasts, and collagen deposition (FIG.36D). The interior of the scaffold was acellular at 2 months and noadverse reaction was seen in the skin. These results show that cryoGelMAgels induce only mild acute inflammation after injection followed by aforeign body reaction.

Example 17: In Vivo Cell Recruitment to Gelatin Cryogels

As a demonstration of the utility of cryoGelMA in vivo, the ability ofthis material to release a chemotactic protein and allow host cellinfiltration into the scaffold was analyzed. GM-CSF, a cytokine involvedin immune cell development that is being widely explored in cancervaccines, was used as a recruitment signal for immune cells (FIG. 37A).Direct incorporation of GM-CSF in the prepolymer solution led to 84±2%encapsulation efficiency after cryopolymerization. GM-CSF is likely tobe released by both diffusion out of the cryogel walls and bydegradation of the walls in vivo. To study the diffusive element of thisprocess, in vitro GM-CSF release over a period of 14 days in DPBScontaining 1% BSA was analyzed (FIGS. 37B-C). Sustained release ofGM-CSF was seen over this period, with an initial burst release followedby a more constant release rate. The total amount of GM-CSF releasedover this period accounted for only ˜2% of the amount encapsulated,indicating that gel degradation mediates payload delivery.

GM-CSF-releasing cryoGelMA was injected subcutaneously to assess cellrecruitment. Implantation of GM-CSF-releasing scaffolds into C57/B16Jmice led to recruitment of ˜20× more live cells at 14 days post-implantrelative to blank cryoGelMA (FIG. 37D). H&E staining showed thin fibrouscapsule formation, and minimal cellular infiltration of blank scaffolds,whereas GM-CSF-releasing gels were surrounded by a thick fibrous capsuleand contained a large primarily granulocytic cellular infiltrate (FIG.37E). These results show that cryoGelMA delivers a chemotactic proteinand allows cell infiltration and recruitment in vivo.

In addition, FIGS. 39A-C depict the massive increase in immune cellrecruitment, a key process in immunotherapy using the devices describedherein, that occurs when GM-CSF is released from gelatin cryogels. Therewas a visible accumulation of cells that occurs in cryoGelMA gels whenGM-CSF is released from the gel (FIG. 39A-B). A comparison of the sizeof recovered blank and GM-CSF releasing cryoGelMA gels 17 days afterinjection into mice shows increased size of the GM-CSF-releasingimplants (FIG. 39C). Histology of center of blank cryoGelMA scaffold 14days after subcutaneous injection in mice shows very few immune cellspresent in the matrix (FIG. 40A). In contrast, histology of center ofGM-CSF releasing cryoGelMA scaffold 14 days after subcutaneous injectionin mice shows massive immune cell recruitment filling the scaffold pores(FIG. 40B). Macrophages (a key antigen presenting cell type) are alsopresent at the site of GM-CSF releasing cryoGelMA scaffold injectionafter 14 days (FIG. 41).

A variety of immune cells are recruited to cryoGelMA at various doses ofGM-CSF. Flow cytometric phenotyping of immune cells residing in blankand GM-CSF releasing gelatin cryogels was performed. Doses of 1, 5, and10 μg of GM-CSF were encapsulated in each cryogel, and cryogels wereimplanted in the flanks of C57/B16J mice for 14 days. Flow cytometricanalysis revealed the presence of roughly the same percentage ofgranulocytes (CD11b+Gr-1+), macrophages (CD11b+F4/80+), dendriticcells/macrophages (CD11c+CD11b+), and CD4+ cells in all conditions.Further staining revealed that a large fraction of CD4+ cells were CD4+regulatory T cells (CD4+/(CD25+FoxP3+)) (FIGS. 42A-F).

Blank cryoGelMA gels created mild inflammation that resolved quickly inthe absence of GM-CSF. Flow cytometric phenotyping of immune cellsresident in gelatin cryogels was performed. Implanted gelatin cryogelswere retrieved at day 6 or day 15 post-injection from C57/B16J mice andanalyzed by flow cytometry for the percentage of macrophage/dendriticcells and NK cells as a percentage of all live cells. A large fractionof resident cells were macrophages/dendritic cells (CD11b+CD11c+) andnatural killer (NK1.1+) cells at day 6, but a significant reduction inthese cells was observed by day 15. Staining for CD4+, CD8+, CD19+, andGr-1+ cells was not substantial at either timepoint.

Example 18: In Vivo Gelatin Cryogel Degradation

The relative degradation of blank- and GM-CSF-cryoGelMA was monitored byin vivo fluorescence imaging of rhodamine-GelMA cryogels.GM-CSF-releasing cryogels degraded much more rapidly than blank cryogelsover the course of 18 weeks (FIG. 38A-B). Since MMPs are thought to bekey players in gelatin degradation, 7 day implanted scaffolds wereassessed for the presence of MMPs using gelatin zymography (FIG. 38C).Degradation bands corresponding to the various molecular weight forms ofMMP-2 and -9, were greatly enhanced in GM-CSF-cryoGelMA relative toblank-cryoGelMA. In vivo imaging using an MMP-sensitive fluorescent dyerevealed MMP activity was concentrated at the implant site of both blankand GM-CSF-releasing cryogels (FIG. 38D). The magnitude of fluorescentsignal from the MMP-sensitive dye at the scaffold site was significantlyenhanced in GM-CSF-releasing scaffolds (FIG. 38E). Collectively, theseresults show that cryoGelMA can be degraded by recruited cells, likelyvia MMP expression.

The results of the studies described herein show that cryoGelMA is acell and tissue compatible biomaterial that can be injected in aminimally invasive manner through a conventional needle. CryoGelMA canbe degraded by MMPs, is capable of controlled release of proteins, andallows cell trafficking within its interconnected pores. Theseadvantageous properties indicate that cryoGelMA is useful forcell-triggered scaffold remodeling and protein release for applicationsin biomaterials-based therapy, e.g., immunotherapy such as cancerimmunotherapy.

Example 19: CpG Incorporation into CryoGelMA Gels

Three incorporation strategies are used for incorporating CpGoligonucleotides into cryoGelMA gels. A slow release profile of CpG fromthe material leads to more sustained immune cell activation, increasingvaccine effectiveness. In one strategy, crosslinked gelatin is mixedwith free CpG (FIG. 44A). Low electrostatic interactions between CpG andgelatin results in rapid release of CpG if directly incorporated intothe gel. A second strategy is to condense the CpG into nanoparticlesusing cationized gelatin and then crosslinking gelatin (e.g.,methacrylated gelatin) will free CpG ODN condensates (FIG. 44B). Thisstrategy is used to slow release of CpG. A third strategy is to usecationized methacrylated gelatin to condense the CpG beforeincorporation (FIG. 45). The methacrylate groups on the resultingparticles allow them to covalently bind to the gel during cryogelformation, localizing the CpG tightly.

Choice of CpG incorporation strategy is employed to tune CpG releaserates for optimal effect, e.g., optimal vaccine efficacy. When 12 ug ofCpG was incorporated into gelatin cryogels, the predicted releaseprofiles were observed, allowing tuning of CpG release for optimalvaccine efficacy (FIG. 46).

Example 20: CryoGelMA is Immune Neutral in the Absence of GM-CSF

Studies were performed to determine the effect of cryoGelMA gels loadedwith or without GM-CSF or CpG on dendritic cell activation. Freshlythawed cryoGelMA gels were vortexed for 1 minute and incubated for 30minutes in cell culture media. Mouse bone marrow derived dendritic cellswere cultured in the conditioned media for 12 hours and analyzed by flowcytometry for activation markers CD40 and CD86. No significantdifference was seen in comparison to cells cultured in untreated culturemedium (FIGS. 47A-B). CryoGelMA conditioned media does not causesubstantial mouse bone marrow derived dendritic cells activation invitro. In contrast, the positive control, lipopolysaccharide (LPS) didcause substantial dendritic cell activation.

The effect of cryoGelMA gels on the production of inflammatory cytokinesby dendritic cells was also assessed. Dendritic cells were seeded oncryoGelMA gels (3D condition). Dendritic cells were also seeded ontissue culture polystyrene (2D condition). LPS was used as a positivecontrol for inducing the production of inflammatory cytokines. CLI is aninhibitor of TLR4, the receptor for endotoxin. See, e.g., Li M. et al.,2006. Mol. Pharmacol., 69:1288-1295. No significant activation ofdendritic cells is seen by culturing dendritic cells on the materialalone (3D) (FIGS. 48A-C).

Thus, cryoGelMA gels do not cause dendritic cell activation in theabsence of GM-CSF or CpG.

Example 21: Seeding of Dendritic Cells in Gelatin Cryogels

CryoGelMA gels were seeded with mouse bone marrow derived dendriticcells. FIG. 49 shows a section of the cryogels containing dendriticcells 48 hours after they were seeded onto the gels.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. An injectable cancer vaccine device comprising a cell-adhesivecryogel composition comprising open interconnected macropores, whereinsaid cryogel composition comprises at least 75% pores, wherein saidcryogel composition is characterized by shape memory followingdeformation by compression through a needle, wherein said cryogelcomposition comprises a crosslinked gelatin polymer or a crosslinkedalginate polymer, and wherein said cryogel composition comprises acancer antigen.
 2. The device of claim 1, wherein said gelatin oralginate is acrylated or methacrylated.
 3. The device of claim 1,wherein the device recruits a cell into the cryogel composition uponinjection into a subject.
 4. The device of claim 3, wherein the cryogelcomposition is degraded by the recruited cell.
 5. The device of claim 1,wherein the cryogel composition is formed by cryopolymerization ofmethacrylated gelatin or methacrylated alginate.
 6. The device of claim5, wherein the cryogel comprises a methacrylated gelatin macromonomerconcentration of 0.5% to 1.4% (w/v).
 7. The device of claim 1, whereinsaid cryogel composition comprises a living attenuated cancer cell. 8.The device of claim 1, wherein said composition comprises a biomoleculein one or more of said open interconnected pores.
 9. The device of claim8, wherein said biomolecule comprises a small molecule, nucleic acid, orprotein.
 10. The device of claim 9, wherein said protein comprisesGM-CSF.
 11. The device of claim 9, wherein said nucleic acid comprises aCpG nucleic acid oligonucleotide (CpG-ODN).
 12. The device of claim 1,wherein the cryogel composition is between 100 μm³ to 100 mm³ in size.13. A method for eliciting an anti-cancer immune response, comprisingadministering to a subject the device of claim
 1. 14. The method ofclaim 13, wherein the device is injected into the subject once to 5times in the lifetime of the subject.
 15. The method of claim 13,wherein the injected device comprises at least 0.5×10⁶ immune cells atleast 1 day after injection into the subject.
 16. The method of claim13, wherein the injected device comprises 10⁷ or fewer cells 15 days ormore after injection.
 17. The method of claim 13, wherein the injecteddevice, a tissue within 10 cm of the injected device, or both comprisesan elevated level of a cytokine compared to the level of the cytokine ata site in the subject more than 10 cm away from the injected device. 18.The method of claim 13, wherein the device increases the survival timeof at least 80% of subjects diagnosed with a cancer by at least 1 monthcompared to the survival time of an untreated subject, wherein increasedsurvival time is determined by comparing the prognosis for survival inthe subject from a time period prior to administration of the device tothe prognosis for survival in the subject following administration ofthe device, wherein an increase in predicted survival time indicatesthat the treatment increased survival of the subject followingadministration of the device.
 19. The method of claim 13, wherein thedevice reduces the rate of tumor growth in the subject compared to therate of tumor growth in an untreated subject.
 20. (canceled)
 21. Themethod of claim 1, wherein the cancer comprises melanoma.
 22. The methodof claim 1, wherein the living attenuated cancer cell comprises anattenuated melanoma cell.
 23. The method of claim 22, wherein theattenuated melanoma cell is an irradiated melanoma cell.
 24. The methodof claim 8, wherein the biomolecule comprises a pathogen-associatedmolecular pattern (PAMP).
 25. The method of claim 8, wherein thebiomolecule comprises a cytokine.
 26. The method of claim 25, whereinthe cytokine comprises Chemokine (C—C motif) ligand 5 (CCL5), eotaxin,interleukin-1alpha (IL-1α), IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9,IL-10, IL-12, IL-13, IL-17, GM-CSF, macrophage inflammatoryprotein-1alpha (MIP-1α), MIP-1β, keratinocyte-derived chemokine (KC),tumor necrosis factor-alpha (TNF-α), granulocyte-colony stimulatingfactor (G-CSF), interferon-alpha (IFN-α), IFN-γ, or monocyte chemotacticprotein-1 (MCP-1).
 27. The device of claim 1, wherein said cryogelcomposition comprises macropores having a diameter of 20 μm to 300 μm.28. The device of claim 1, wherein said cryogel composition ischaracterized by shape memory following deformation by compressionthrough a needle, such that said cryogel returns to its originalundeformed three-dimensional shape less than one second aftercompression through the needle.
 29. The device of claim 1, wherein saidhighly crosslinked cryogel composition comprises a crosslinking densityof at least 50% polymer crosslinking.
 30. The device of claim 1, whereinsaid highly crosslinked cryogel composition comprises a crosslinkingdensity of 50-100% polymer crosslinking.
 31. The device of claim 1,further comprising microspheres.
 32. The device of claim 31, whereinsaid microspheres comprise PLGA.
 33. The device of claim 31, whereinsaid microspheres are physically entrapped within the cryogelcomposition.
 34. The device of claim 31, wherein said microspherescomprise GM-CSF.
 35. The device of claim 31, wherein said microspherescomprise a drug.
 36. A syringe comprising (i) a needle; (ii) a reservoircomprising the device of claim 1; and (iii) a plunger.
 37. The syringeof claim 36, comprising a 16-gauge, an 18-gauge, a 22-gauge, a 24-gauge,a 26-gauge, a 28-gauge, a 30-gauge, a 32-gauge, or a 34-gauge needle.38. The syringe of claim 36, comprising an 18 to 30-gauge needle. 39.The syringe of claim 38, wherein the device is between 1 mm³ to 50 mm³in size.
 40. The syringe of claim 36, wherein the device comprises alive attenuated cancer cell, and wherein 90% or more of the liveattenuated cancer cells survive passage of the device through theneedle.
 41. The device of claim 1, further comprising magneticparticles.
 42. The device of claim 41, wherein said magnetic particlescomprise Fe₃O₄ nanoparticles or Fe₃O₄ micro-particles.
 43. The device ofclaim 1, further comprising mechanophore-molecules.
 44. The device ofclaim 43, wherein said mechanophore molecules comprise polydiacetyleneliposomes.
 45. The device of claim 1, wherein the cancer comprisesmelanoma, breast cancer, central nervous system cancer, lung cancer,leukemia, multiple myeloma, renal cancer, malignant glioma,medulloblastoma, colon cancer, stomach cancer, sarcoma, cervical cancer,ovarian cancer, lymphoma, Non-Hodgkin's lymphoma, pancreatic cancer,prostate cancer, thyroid cancer, rectal cancer, endometrial cancer, orbladder cancer.